Multivalent binding composition for nucleic acid analysis

ABSTRACT

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

CROSS-REFERENCE

This application is a continuation of International Patent ApplicationNo. PCT/US2020/052305, filed Sep. 23, 2020, which claims the benefit ofU.S. Provisional Application No. 62/904,623, filed on Sep. 23, 2019,each of which is incorporated by reference in its entirety.

BACKGROUND

Emerging methods of diagnosis for cancers, infectious diseases,dysbiosis, and other disease and conditions rely on next generationsequencing (NGS) methods to provide high resolution genetic and genomicdata, enabling robust and personalized diagnosis, treatment planning,and eventually cures for diseases that were not previously tractable.While powerful, NGS methods are still limited by the methods availableto provide nucleic acid samples to the instruments that carry out theactual sequencing. For example, identifying the precise nature of themutations present in a particular tumor requires isolation of tumortissue, isolation of nucleic acids, and multiple steps in thepreparation of samples for particular sequencing methods, prior to theengagement of the instrument to obtain actual sequence data.Additionally, deconvolution and processing of sequence data in a waythat allows the correlation of particular sequences with particularcells or tissues is complicated by the nature of NGS technologies, whichoften require pooling of samples, during which spatial and cellularidentity information is lost.

Various methods have been proposed to address this problem of the lossof cellular addressability in NGS methods, toward the goal of providingmolecular diagnostics with higher spatial or tissue resolution. Forexample, some approaches rely on separation of cells, followed byapplying unique barcodes to the nucleic acids from each individual cell,and then bulk sequencing, using the unique barcodes to identify thesequences associated with each individual cell after the sequencing runis complete. This can be achieved, for example, by exposing individualcells to lysis and hybridization mixtures within an isolated environmentsuch as a bead or emulsion. These methods may further require enrichmentor processing of the target cell subpopulation, such as by cell sortingfor circulating cells, or by tissue harvesting followed by dissociationand protease treatment for solid tumor cells.

While such methods can obtain cellularly addressable information, theyface severe limitations, such as difficulties in processing solidtissues, and throughput rates limited by the ability to isolate, tag,and prepare nucleic acids for sequencing. Likewise, there arelimitations associated with the need to transfer prepared libraries toseparate instruments, systems, or locations in order to carry outsequencing steps. This provides a practical limitation on sequencingthroughput of approximately 50,000 cells per sequencing run, which,given the vastly larger number of cells present in a diagnosticallyrelevant sample of a tissue, secretion, excretion, or exudate, or amicrobiome sample, places strict limits on the sensitivity and utilityof these assays. A level of addressability may be achieved simply byphysically isolating samples and performing isolations, librarypreparation, and sequencing reactions in a known sequence. However, thisprocess is labor intensive and time consuming, making it impractical asa means of screening large numbers of patients or as a means ofdeploying systematic screening methods.

Accordingly, there is a need for compositions and methods that canincrease the accuracy and throughput of cellularly addressablesequencing methods, as well as cellularly or spatially addressablesequencing methods that obviate the aforementioned limitations ofexisting technologies.

SUMMARY

Aspects disclosed herein provide methods for analyzing a target nucleicacid sequence, the method comprising: (a) providing a plurality ofprimed nucleic acid molecules; (b) contacting said plurality of primednucleic acid molecules with a detectable polymer-nucleotide conjugateunder conditions suitable to form a binding complex between a nucleotidemoiety of said detectable polymer-nucleotide conjugate and a nucleotideof a primed nucleic acid molecule of said plurality of primed nucleicacid molecules; (c) detecting said binding complex; and (d) performing(b) to (c) for nucleotides in said primed nucleic acid molecule, therebyidentifying a sequence of said primed nucleic acid molecule. In someembodiments, performing (b) to (d) is performed in less than or equal toabout 60 minutes. In some embodiments, performing (b) to (d) isperformed in less than or equal to about 30 minutes. In someembodiments, the detectable polymer-nucleotide conjugate comprises aplurality of detectable polymer-nucleotide conjugates, wherein each ofthe plurality of detectable polymer-nucleotide conjugate comprise adifferent type of nucleotide moiety. In some embodiments, said pluralityof nucleic acid molecules is coupled to an interior surface of a flowcell. In some embodiments, said interior surface of said flow cellcomprises one or more hydrophilic polymer layers. In some embodiments,said one or more hydrophilic polymer layers comprises a polymercomprising polyethylene glycol (PEG). In some embodiments, said one ormore hydrophilic polymer layers comprises a branched polymer.

Aspects disclosed herein provide methods for analyzing a biologicalsample comprising: (a) detecting a multivalent binding complex formed ina presence of a biological sample or derivative thereof between a targetnucleic acid sequence of a target nucleic acid molecule or derivativethereof and a detectable polymer-nucleotide conjugate; and (b)determining an origin of said target nucleic acid sequence in saidbiological sample or derivative thereof. In some embodiments,determining in (b) is performed at least in part by analyzing a relativethree-dimensional relationship between said target nucleic acid sequenceand a point of reference of said biological sample or derivativethereof. In some embodiments, methods further comprise contacting saidbiological sample or derivative thereof with said detectablepolymer-nucleotide conjugate in said presence of the biological sample.In some embodiments, methods further comprise coupling at least aportion of said target nucleic acid sequence to a captureoligonucleotide molecule coupled to a surface of a substrate. In someembodiments, said surface has a water contact angle of less than orequal to 45 degrees. In some embodiments, coupling comprises hybridizingin a presence of a hybridization buffer comprising: (i) a first polaraprotic solvent having a dielectric constant that is no greater than 40and having a polarity index of 4-9; and (ii) a second polar aproticsolvent having a dielectric constant that is less than or equal to 115.In some embodiments, methods further comprise immobilizing saidbiological sample or derivative thereof on said surface in a manner thatis sufficient to fix said relative three-dimensional relationship. Insome embodiments, methods further comprise amplifying said targetnucleic acid sequence on said surface of said substrate, optionally,using rolling circle amplification. In some embodiments, an image ofsaid surface in said presence of said biological sample or derivativethereof exhibits a contrast-to-noise ratio of greater than or equal toabout 5 as measured by: (a) contacting said surface with a fluorescentlylabeled nucleotide molecule comprising a nucleic acid sequence that iscomplementary to at least a portion of a capture oligonucleotideimmobilized to said surface; and (b) following (a), imaging said surfaceusing an inverted microscope and a camera under non-signal saturatingconditions while said surface is immersed in a buffer. In someembodiments, methods further comprise performing a nucleotide bindingreaction between a nucleotide moiety coupled to said polymer-nucleotideconjugate and said target nucleic acid molecule or derivative thereof.In some embodiments, said target nucleic acid molecule or derivativethereof is a deoxyribonucleic acid (DNA) molecule. In some embodiments,said biological sample or derivative thereof comprises a fluidbiological sample. In some embodiments, said origin is a canceroustissue.

Aspects disclosed herein provide methods for identifying at least aportion of a sub-cellular component within a cell or tissue in situ, themethod comprising: (a) detecting a signal from a multivalent bindingcomplex between said sub-cellular component or derivative thereof and adetectable polymer-nucleotide conjugate; and (b) processing at leastsaid signal detected in (a) to identify said at least said portion ofsaid sub-cellular component or derivative thereof. In some embodiments,said sub-cellular component or derivative thereof is a nucleic acid. Insome embodiments, said nucleic acid is DNA. In some embodiments, methodsfurther comprise: (c) immobilizing said cell or said tissue on a surfaceof a substrate. In some embodiments, methods further comprise: (d)coupling at least a portion of said sub-cellular component to a capturemolecule coupled to a said surface. In some embodiments, methods furthercomprise: (e) permeabilizing said tissue or lysing said cell prior todetecting in (a). In some embodiments, said surface has a water contactangle of less than or equal to 45 degrees. In some embodiments, couplingin (d) comprises hybridizing said capture molecule with said at leastsaid portion of said sub-cellular component in a presence of ahybridization buffer comprising: (i) a first polar aprotic solventhaving a dielectric constant that is no greater than 40 and having apolarity index of 4-9; and (ii) a second polar aprotic solvent having adielectric constant that is less than or equal to 115. In someembodiments, an image of said surface exhibits a contrast-to-noise ratioof greater than or equal to about 5 as measured by: (a) contacting saidsurface with a fluorescently labeled nucleotide molecule comprising anucleic acid sequence that is complementary to at least a portion of acapture oligonucleotide immobilized to said surface; and (b) following(a), imaging said surface using an inverted microscope and a cameraunder non-signal saturating conditions while said surface is immersed ina buffer. In some embodiments, detecting said signal from saidmultivalent binding complex in (a) comprises performing a nucleotidebinding reaction between a nucleotide moiety coupled to saidpolymer-nucleotide conjugate and said sub-cellular component orderivative thereof. In some embodiments, said tissue is from a tumor.

Aspects disclosed herein provide systems for analyzing a biologicalsample comprising: a substrate comprising a surface having coupledthereto a polymer layer suitable to immobilize said biological sample tosaid surface, wherein: said biological sample or derivative thereofcomprises a target nucleic acid molecule or derivative thereof; saidpolymer layer is configured to couple with (i) said biological sample orderivative thereof, or (ii) said target nucleic acid molecule orderivative thereof; said target nucleic acid molecule or derivativethereof is configured to couple with a nucleotide moiety comprising adetectable label; and an image of said surface exhibits acontrast-to-noise ratio of greater than or equal to about 5 when saidimage of said surface is obtained using an inverted microscope and acamera under non-signal saturating conditions while said surface isimmersed in a buffer and wherein said detectable label is a fluorescentdye. In some embodiments, said polymer layer is hydrophilic. In someembodiments, systems further comprise a fixing agent that fixes saidbiological sample to said surface when said biological sample iscontacted with said fixing agent while adjacent to said surface. In someembodiments, said fixing agent comprises formaldehyde or glutaraldehyde.In some embodiments, said target nucleic acid molecule is a concatemer.In some embodiments, said target nucleic acid molecule comprises auniversal sequence region comprising a spatial barcode sequence or asample barcode sequence configured to retain an origin of said targetnucleic acid molecule in said biological sample. In some embodiments, animage of said surface exhibits a contrast-to-noise ratio of greater thanor equal to about 10 when said image of said surface is obtained. Insome embodiments, said substrate is a flow cell device comprising afirst flow channel and, optionally, a second flow channel. In someembodiments, said substrate is a planar substrate that is reflective,transparent, or translucent. In some embodiments, said flow cell deviceis a capillary flow cell device.

Aspects disclosed herein provide systems for analyzing nucleic acidsequence information in a biological sample or derivative thereof, thesystem comprising: one or more computer processors programed to: (a)detect a signal from a multivalent binding complex formed in a presenceof said biological sample or derivative thereof between a target nucleicacid sequence of a target nucleic acid molecule or derivative thereofand a detectable polymer-nucleotide conjugate, wherein said signal isindicative of an identity of a nucleotide in said target nucleic acidsequence; and (b) determine an origin of said target nucleic acidsequence in said biological sample. In some embodiments, said one ormore computer processors is programed to determine said origin of saidtarget nucleic acid sequence in (b) by analyzing a relativethree-dimensional relationship between said target nucleic acid moleculeor derivative thereof and said biological sample or derivative thereof.In some embodiments, said system further comprises a database configuredto store three-dimensional data related to said origin of said targetnucleic acid sequence. In some embodiments, said database is furtherconfigured to store sequencing data comprising said identity of saidnucleotide in said target nucleic acid sequence. In some embodiments,(b) is performed by associating said sequencing data and saidthree-dimensional data. In some embodiments, said one or more computerprocessors is programed to identify said target nucleic acid sequence inless than 60 minutes by repeating (a) to (b). In some embodiments, saidone or more computer processors is programed to perform (a) to (b) withan accuracy of base-calling that is characterized by a Q-score ofgreater than 25 for at least 80% of nucleotides identified. In someembodiments, said detectable polymer-nucleotide conjugate comprises: (a)a polymer core; and (b) two or more nucleotide moieties attached to saidpolymer core, wherein said polymer-nucleotide conjugate is configured toform a multivalent binding complex between said two or more nucleotidemoieties and said target nucleic acid molecule or derivative thereof. Insome embodiments, said one or more nucleotide moieties comprises anucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog.In some embodiments, said polymer core comprises a polymer that has astar, comb, cross-linked, bottle brush, or dendrimer configuration. Insome embodiments, said polymer core comprises a branched polyethyleneglycol (PEG) molecule. In some embodiments, systems further comprise anoptical imaging system comprising a field-of-view (FOV) greater than 1.0mm².

Aspects disclosed herein provide kits comprising: (a) a detectablepolymer-nucleotide conjugate comprising: (i) a polymer core; and (ii)(ii) two or more nucleotide moieties attached to said polymer core; and(b) instructions for identifying at least a portion of a sub-cellularcomponent within a cell or tissue in situ by contacting said detectablepolymer-nucleotide conjugate with said sub-cellular component underconditions sufficient to form a multivalent binding complex between saidtwo or more nucleotide moieties and said sub-cellular component. In someembodiments, kits comprise 4 types of said detectable polymer-nucleotideconjugate, wherein each of said 4 types has a different nucleotidemoiety attached thereto.

Aspects disclosed herein comprise kits comprising: (a) a substratecomprising a surface having coupled thereto a polymer layer suitable toimmobilize a biological sample or derivative thereof to said surface;and (b) instructions for determining a target nucleic acid sequence andan origin of said target nucleic acid sequence in said biological sampleor derivative on said surface. In some embodiments, kits furthercomprise: (a) a hybridization buffer comprising: (i) a first polaraprotic solvent having a dielectric constant that is no greater than 40and having a polarity index of 4-9; and (ii) a second polar aproticsolvent having a dielectric constant that is less than or equal to 115;and (b) instructions for hybridizing at least a portion of said targetnucleic acid sequence to at least a portion of a capture oligonucleotidecoupled to said surface.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic illustration of one embodiment of the low bindingsupport comprising a glass substrate and alternating layers ofhydrophilic coatings which are covalently or non-covalently adhered tothe glass, and which further comprises chemically-reactive functionalgroups that serve as attachment sites for oligonucleotide primers (e.g.,capture oligonucleotides and circularization oligonucleotides) accordingto an embodiment of the present disclosure. In an alternativeembodiment, the support can be made of any material such as glass,plastic or a polymer material.

FIG. 2 is a schematic showing a support comprising a captureoligonucleotide and a circularization oligonucleotide immobilizedthereon according to an embodiment of the present disclosure. In someembodiments, the support comprises a plurality of captureoligonucleotides and a plurality of circularization oligonucleotidesimmobilized thereon.

FIG. 3 is a schematic showing a support comprising a plurality ofcapture oligonucleotides and circularization oligonucleotidesimmobilized thereon and a biological sample (e.g., a tissue sample)placed on the support (see the left schematic), according to anembodiment of the present disclosure. FIG. 3 shows an enlarged sectionof the support having an array of features each having a circular shapeand labeled for spatial identification on the support (see the rightschematic). Each feature comprises a plurality of immobilized captureoligonucleotides and circularization oligonucleotides.

FIG. 4 is a schematic showing a support comprising a captureoligonucleotide immobilized thereon, and a soluble circularizationoligonucleotide, according to an embodiment of the present disclosure.In some embodiments, the support comprises a plurality of captureoligonucleotides immobilized thereon.

FIG. 5A is a schematic showing a nucleotide arm of a polymer-nucleotideconjugate according to an embodiment of the present disclosure.

FIG. 5B is a schematic of a polymer-nucleotide conjugate comprising acore attached to a plurality of nucleotide arms where each nucleotidearm comprises (i) a core attachment moiety, (ii) a spacer, (iii) alinker, and (iv) a nucleotide unit, according to an embodiment of thepresent disclosure.

FIG. 5C is a schematic of a polymer-nucleotide conjugate, in dendrimerform, comprising a branched polymer which radiates from a centralattachment point or central moiety, where a plurality of nucleotide armsradiate from the central attachment point, according to an embodiment ofthe present disclosure.

FIG. 5D is a nucleotide arm of a polymer-nucleotide conjugate comprisinga biotin core attachment moiety, a spacer, an aliphatic chain linker,and a nucleotide attached to the linker via a propargyl link at thebase, according to an embodiment of the present disclosure.

FIG. 6A shows structures of a spacer and linkers of a polymer-nucleotideconjugate according to an embodiment of the present disclosure.

FIG. 6B-6C shows structures of additional linkers of apolymer-nucleotide conjugate according to an embodiment of the presentdisclosure.

FIG. 7 shows a work flow according to an embodiment of the presentdisclosure.

FIGS. 8A-8B schematically illustrate non-limiting examples of imagingdual surface support structures for presenting sample sites for imagingby the imaging systems disclosed herein. FIG. 8A: illustration ofimaging front and rear interior surfaces of a flow cell. FIG. 8B:illustration of imaging front and rear exterior surfaces of a substrate.

FIGS. 9A-9B illustrate a non-limiting example of a multi-channelfluorescence imaging module comprising a dichroic beam splitter fortransmitting an excitation light beam to a sample, and for receiving andredirecting by reflection the resultant fluorescence emission to fourdetection channels configured for detection of fluorescence emission atfour different respective wavelengths or wavelength bands. FIG. 9A: topisometric view. FIG. 9B: bottom isometric view.

FIGS. 10A-10B illustrate the optical paths within the multi-channelfluorescence imaging module of FIGS. 10A and 10B comprising a dichroicbeam splitter for transmitting an excitation light beam to a sample, andfor receiving and redirecting by reflection a resultant fluorescenceemission to four detection channels for detection of fluorescenceemission at four different respective wavelengths or wavelength bands.FIG. 10A: top view. FIG. 10B: side view.

FIGS. 11A-11B illustrate the modulation transfer function (MTF) of anexample dual surface imaging system disclosed herein having a numericalaperture (NA) of 0.3. FIG. 11A: first surface. FIG. 11B: second surface.

FIGS. 12A-12B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.5. FIG. 12A: first surface.FIG. 12B: second surface.

FIGS. 13A-13B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.7. FIG. 13A: first surface.FIG. 15B: second surface.

FIGS. 14A-14B provide plots of the calculated Strehl ratio for imaging asecond flow cell surface through a first flow cell surface. FIG. 14A:plot of the Strehl ratios for imaging a second flow cell surface througha first flow cell surface as a function of the thickness of theintervening fluid layer (fluid channel height) for different objectivelens and/or optical system numerical apertures. FIG. 14B: plot of theStrehl ratio as a function of numerical aperture for imaging a secondflow cell surface through a first flow cell surface and an interveninglayer of water having a thickness of 0.1 mm.

FIG. 15 provides an optical ray tracing diagram for an objective lensdesign that has been designed for imaging a surface on the opposite sideof a 0.17 mm thick coverslip.

FIG. 16 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.17 mm thickcoverslip.

FIG. 17 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.3 mm thickcoverslip.

FIG. 18 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid.

FIG. 19 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface on the opposite side of a 1.0 mm thickcoverslip.

FIG. 20 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid.

FIG. 21 provides a ray tracing diagram for a tube lens design which, ifused in conjunction with the objective lens illustrated in FIG. 15,provides for improved dual-side imaging through a 1 mm thick coverslip.

FIG. 22 provides a plot of the modulation transfer function for thecombination of objective lens and tube lens illustrated in FIG. 15 as afunction of spatial frequency when used to image a surface on theopposite side of a 1.0 mm thick coverslip.

FIG. 23 provides a plot of the modulation transfer function for thecombination of objective lens and tube lens illustrated in FIG. 15 as afunction of spatial frequency when used to image a surface that isseparated from that on the opposite side of a 1.0 mm thick coverslip bya 0.1 mm thick layer of aqueous fluid.

FIG. 24 illustrates one non-limiting example of a single capillary flowcell having 2 fluidic adaptors.

FIG. 25 illustrates one non-limiting example of a flow cell cartridgecomprising a chassis, fluidic adapters, and optionally other components,that is designed to hold two capillaries.

FIG. 26 illustrates one non-limiting example of a system comprising asingle capillary flow cell connected to various fluid flow controlcomponents, where the single capillary is compatible with mounting on amicroscope stage or in a custom imaging instrument for use in variousimaging applications.

FIG. 27 is a schematic showing a support having immobilized thereon acapture oligonucleotide and circularization oligonucleotide, and anexemplary method for capturing nucleic acids from a cellular biologicalsample which is positioned on the support, according to variousembodiments described herein.

FIG. 28 is a schematic showing a support having immobilized thereon acapture oligonucleotide, and an exemplary method for capturing nucleicacids from a cellular biological sample which is positioned on thesupport where the method includes use of a soluble circularizationoligonucleotide, according to various embodiments described herein.

DETAILED DESCRIPTION

Provided herein are spatially addressable and cellularly addressablesequencing methods and systems, as well as compositions, devices, andkits useful for performing the methods and systems described herein. Themethods and systems described herein may utilize a polymer-nucleotideconjugate in a nucleotide binding reaction in situ. The nucleotidebinding reaction may be performed on a hydrophilic surface, whichprovide a number of advantages described herein. Hybridization buffersthat comprise polar and aprotic solvents in combination with a pH bufferare also provided herein. Also provided are optical systems useful forspatially resolving sequencing data. In some embodiments, the opticalsystems described herein have a field of view that is greater than 1.0mm².

As shown in FIG. 7, methods described herein comprise, in someembodiments: (a) providing a surface (e.g., low non-specific bindingsurface) having a plurality of capture oligonucleotides coupled thereto(22); fixing a biological sample containing a target nucleic acidmolecule to the surface, and optionally permeabilizing the biologicalsample (23); (c) contacting the plurality of capture oligonucleotideswith the target nucleic acid molecule under conditions sufficient toallow hybridization of at least a portion of the plurality of captureoligonucleotides to the target nucleic acid molecule (24); (d)amplifying the target nucleic acid molecule to produce amplified targetnucleic acid molecules or derivatives thereof (25); (e) contacting theamplified target nucleic acid molecules or derivatives thereof with oneor more polymerases and one or more primer nucleic acid molecules havinga primer sequence that is complementary to one or more regions of theamplified target nucleic acid molecules or derivatives thereof, toproduce primed target nucleic acid molecules or derivatives thereof(26); (f) contacting the primed target nucleic acid molecules orderivatives thereof with a polymer-nucleotide conjugate comprising twoor more nucleotide moieties coupled to a polymer (e.g., PEG) core thatis labeled with a detectable label (e.g., fluorophore) (27); (g)detecting a multivalent binding complex formed between the primed targetnucleic acid molecules or derivatives thereof and the polymer-nucleotideconjugate (28); (h) wash the surface with a buffer sufficient to removethe polymer-nucleotide conjugate from the primed target nucleic acidmolecule or derivative thereof (29); (i) incorporate a nucleotide thatdoes not contain a detectable label and which optionally comprises ablocking group (e.g., azidomethyl) that blocks incorporation of a secondnucleotide at an N+1 position on the primed target nucleic acid moleculeor derivative thereof (30); and (j) optionally, repeat steps (f)-(j)(31).

Existing methods of spatially addressable sequence identification (alsoreferred to herein as spatial transcriptomic technology) suffer from lowsensitivity, non-specificity and inaccurate spatial location of thetranscripts of interest. In contrast, the methods, systems, compositionsand kits described herein overcome these challenges, for example, byleveraging low non-specific binding surfaces, high efficiencyhybridization buffers, methods to prepare nanoballs with high copynumber, and multivalent molecules.

The low non-specific binding and improved signal of the instantdisclosure provide significantly improved contrast-to-noise (CNR)ratios, as compared with existing methodologies. The CNR is at leastpartially improved by utilizing highly compact foci of reaction (e.g.,highly compact nucleic acid clusters with high copy number), highlyefficient surface hybridization (allowing precise localization ofnucleic acid capture), and very low background, while enabling highlyefficient capture, amplification, and clustering of target nucleicacids. When a biological sample (e.g., tissue, cellular suspension) iscoupled to the substrate, the sequencing reaction can be performed inthe presence of the biological sample. Analysis of the sequencingreaction can be performed in a manner that provides cellularaddressability and/or spatial addressability, such that sequence datamay be linked to the tissue, cell type, physiological location, orspatial location from which it was derived.

The high efficiency hybridization buffers described herein promote highstringency (e.g., specificity), speed, and efficacy of nucleic acidhybridization reactions and increases the efficiency of the subsequentamplification and sequencing steps. The high efficiency hybridizationbuffers can significantly shorten nucleic acid hybridization times, anddecreases sample input requirements. The high efficiency hybridizationbuffers can be used for nucleic acid annealing workflows at isothermalconditions which eliminates requirement of a cooling step for annealing.The high efficiency hybridization buffers provide precise localizationof nucleic acid capture on a surface for accurate spatial localizationof nucleic acids (e.g., transcripts) that originate from a cell ortissue.

The rolling circle amplification methods described herein includes atwo-stage method that employs non-catalytic and then catalytic divalentcations to synchronize the rolling circle amplification events on asurface and generate concatemers. The rolling circle amplificationreaction can be followed by a relaxant condition and a flexingamplification reaction which generates new concatemers from the existingconcatemers. Together, these amplification methods generate highlycompact nanoballs containing high copy number of the target sequencewhich improves sequencing signal intensity.

The nucleic acid analysis methods described herein may have higherthroughput than existing methods, allowing the analysis of 50,000,100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000 or more cells perrun, enabling vastly higher diagnostic sensitivity by allowing thedetection of, in principal, mutations in as few as one cell per million.A further advantage of the nucleic acid methods disclosed herein is thatthe reactions required may be carried out at a single temperature (e.g.,isothermal conditions), such as, for example, 20° C., 25° C., 30° C.,35° C., 37° C., 40° C., 42° C., 50° C., 60° C., 65° C., 70° C., or 72°C. or more, or within a range defined by any two of the foregoing.

The multivalent molecules used during the sequencing reaction offer manyadvantages that are not provided by free nucleotides. The multivalentmolecules comprise a core attached to multiple arms with each armtethered to a nucleotide. The multivalent molecules increase the localconcentration of nucleotides in proximity of a polymerase/templatebinding site. The multivalent molecules also exhibit increasedpersistence time in formation of a stable ternary complex with apolymerase and nucleic acid template. Thus, a labeled multivalentmolecule provides shorter imaging time and increase signal intensityduring a sequencing reaction.

Cellular and spatial resolution of sequencing data generating using themethods and systems described herein are achieved by the imaging methodsand systems described herein, which provide increased optical resolutionand improved image quality for genomics applications.

Disclosed herein are optical component and system designs forhigh-performance fluorescence imaging methods and systems that mayprovide any one or more of: larger fields-of-view, improved opticalresolution (including high performance optical resolution), improvedcontrast, improved image quality, faster transitions between imagecapture when repositioning the sample plane to capture a series ofimages (e.g., of different fields-of-view), improved imaging system dutycycle, and higher throughput image acquisition and analysis.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications comprising the use of thickflow cell walls (e.g., wall (or coverslip) thickness >700 μm) and fluidchannels (e.g., fluid channel height or thickness of 50-200 μm) may beachieved using novel objective lens designs that correct for opticalaberration introduced by imaging surfaces on the opposite side of thickcoverslips and/or fluid channels from the objective.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications comprising the use of thickflow cell walls (e.g., wall (or coverslip) thickness >700 μm) and fluidchannels (e.g., fluid channel height or thickness of 50-200 μm) may beachieved even when using commercially-available, off-the-shelfobjectives by using a novel tube lens design that, unlike the tube lensin a conventional microscope that simply forms an image at theintermediate image plane, corrects for the optical aberrations inducedby the thick flow cell walls and/or intervening fluid layer incombination with the objective.

In some instances, improvements in imaging performance, e.g., formultichannel (e.g., two-color or four-color) imaging applications, maybe achieved by using multiple tube lenses, one for each imaging channel,where each tube lens design has been optimized for the specificwavelength range used in that imaging channel.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications, may be achieved by using anelectro-optical phase plate in combination with an objective lens tocompensate for the optical aberrations induced by the layer of fluidseparating the upper (near) and lower (far) interior surfaces of a flowcell. In some instances, this design approach may also compensate forvibrations introduced by, e.g., a motion-actuated compensator that ismoved in or out of the optical path depending on which surface of theflow cell is being imaged.

Further advantageous features of the disclosed imaging optics designsmay include the position and orientation of one or more excitation lightsources and one or more detection optical paths with respect to theobjective lens and to a dichroic filter that receives the excitationbeam. The excitation beam may also be linearly-polarized and theorientation of the linear polarization may be such that s-polarizedlight is incident on the dichroic reflective surface of the dichroicfilter. Such features may potentially improve excitation beam filteringand/or reduce wave front error introduced into the emission light beamdue to, e.g., surface deformation of dichroic filters.

Although discussed herein primarily in the context of fluorescenceimaging (including, e.g., fluorescence microscopy imaging, fluorescenceconfocal imaging, two-photon fluorescence, and the like), it will beunderstood by those of skill in the art that many of the disclosedoptical design approaches and features are applicable to other imagingmodes, e.g., bright-field imaging, dark-field imaging, phase contrastimaging, and the like.

In addition to the optical components and imaging system designsdisclosed herein, flow cell devices and systems for performing a varietyof genomic analysis methods, including cellularly-addressable nucleicacid sequencing, are disclosed that may comprise various combinations ofthe disclosed optical, mechanical, fluidic, thermal, electrical, andcomputing modules or sub-systems. The advantages conferred by thedisclosed flow cell devices, cartridges, and analysis systems include,but are not limited to: (i) reduced device and system manufacturingcomplexity and cost, (ii) significantly lower consumable costs (e.g., ascompared to those for currently available nucleic acid sequencingsystems), (iii) compatibility with typical flow cell surfacefunctionalization methods, (iv) flexible flow control when combined withmicrofluidic components, e.g., syringe pumps and diaphragm valves, etc.,and (v) flexible system throughput.

In some instances, the disclosed capillary flow-cell devices andcapillary flow cell cartridges may be constructed from off-the-shelf,disposable, single lumen (e.g., single fluid flow channel) ormulti-lumen capillaries that may also comprise fluidic adaptors,cartridge chassis, one or more integrated fluid flow control components,or any combination thereof. In some instances, the disclosed flowcell-based systems that may comprise one or more capillary flow celldevices (or microfluidic chips), one or more capillary flow cellcartridges (or microfluidic cartridges), fluid flow controller modules,temperature control modules, imaging modules, or any combinationthereof. The design features of some disclosed capillary flow celldevices, cartridges, and systems include, but are not limited to, (i)unitary flow channel construction, (ii) sealed, reliable, and repetitiveswitching between reagent flows that can be implemented with a simpleload/unload mechanism such that fluidic interfaces between the systemand capillaries are reliably sealed, thereby facilitating capillaryreplacement and system reuse, and enabling precise control of reactionconditions such as reagent concentration, pH, and temperature, (iii)replaceable single fluid flow channel devices or capillary flow cellcartridges comprising multiple flow channels that can be usedinterchangeably to provide flexible system throughput, and (iv)compatibility with a wide variety of detection methods such asfluorescence imaging.

Although the disclosed capillary flow cell and microfluidic devices andsystems are described primarily in the context of their use for nucleicacid sequencing applications, various aspects of the disclosed devicesand systems may be applied not only to nucleic acid sequencing but alsoto any other type of chemical analysis, biochemical analysis, nucleicacid analysis, cell analysis, or tissue analysis application. It shallbe understood that different aspects of the disclosed methods, devices,and systems can be appreciated individually, collectively, or incombination with each other.

Embodiments described herein provide significant advantages for thediagnosis of cancers, including both circulating and solid tumors, theanalysis of biopsy samples, e.g., for the diagnosis of geneticdisorders, the analysis of microbiome samples, e.g., for the diagnosisof disorders linked to dysbiosis in microbial flora, for the diagnosisof disorders accompanying a secretion or exudate, or for the assessmentof general health or disease risk, where such risk may be assessed withrespect to the presence or identity of particular genetic sequences in aparticular cell, tissue, or location. For example, it may be useful touse high resolution cellularly addressable sequencing techniques toidentify the presence of low levels of circulating tumor cells for thediagnosis of blood cancers or early metastases.

In some embodiments, cells in a tissue or individual cells may beexposed to a surface under conditions optimized for binding (capturing)of target nucleic acids by, for example, inclusion of high densities ofpoly-T or poly-dT oligonucleotides for the capture of RNA transcriptsfollowed by reverse transcription, or inclusion of random-sequencecapture oligonucleotides for hybridization to genomic, circulating, ororganellar DNA. In some embodiments, this capture process may befollowed by one or more library preparation steps, such as appending atleast one adaptor to the captured nucleic acid where the adaptor caninclude an index sequence, barcode sequence and/or a Unique MolecularIdentifier (UMI). The adaptor appending step can be conducted byligation (e.g., blunt end ligation) or by use of “splint”oligonucleotides. These library preparation steps may result in or mayfurther include circularization of the captured nucleic acids. In someembodiments, a circularized nucleic acid molecule, may be amplified suchas by Rolling Circle Amplification (RCA), yielding a large multicopynucleic acid molecule (e.g., concatemer) comprising multiple tandemrepeat sequences of the target sequence. In some embodiments, said largemulticopy nucleic acid may form a condensed state, such as by the use ofbuffer conditions favoring compact DNA states, surfaces having highdensities of capture oligonucleotides, the use of bivalent or bispecificoligonucleotides that bridge two or more sites within a large multicopynucleic acid (“clustering oligonucleotides” or “clustering oligos”), orby any combination of the foregoing, or by any method as is or becomesknown in the art to produce compact clusters comprising large multicopynucleic acids.

In some embodiments, the surface used to capture nucleic acids from thetissue or cells may be composed to retain nucleic acids with highactivity while simultaneously maintaining a low level of binding forunwanted proteins, lipids, carbohydrates, or other components of celldebris. Thus, the surfaces contemplated herein are capable of binding tothe nucleic acids from cells in a tissue, or from a single cell, thatis/are lysed in contact with or in proximity to the surface. Further,the surfaces do not retain cell debris, nor do they show significantnonspecific binding of added proteins such as nucleic acid polymerases,or other molecules, moieties, particles, or items such as dye moleculesor fluorophores.

In some embodiments, cell lysis (and optionally nucleic acidfragmentation) are carried out in contact with or in proximity to thesurface such that a significant amount, such as a representativequantity, or substantially all, of the DNA, RNA, or other target nucleicacids released from the cell or tissue sample will be captured by thesurface. The surface may be composed such that cells can be flowed overthe surface in order to reach capture sites on said surface.Alternatively, a capture surface may be composed such that a tissue(e.g., tissue section) can be placed in contact or in fluidcommunication with the surface, where reagents may then be flowed overthe tissue in such a manner as to facilitate the capture in situ ofnucleic acids from the tissue, such that the nucleic acids from one cellor region of the tissue will be captured in the same location andorientation relative to the nucleic acids from other cells or regions ofthe tissue, as the nucleic acids were oriented or located within theintact tissue.

In some embodiments, the capturing, adaptor-appending, circularizing,amplifying, and clustering of the target nucleic acids can be carriedout while attached to, or in close proximity to, the surface.Alternatively, one or more of the foregoing preparatory steps may becarried out in free solution, or while attached to beads.

Spatially resolved binding of a cell-specific nucleic acid complementsuch as, for example, a cellular genome or a cellular transcriptome,followed by adaptor-appending, circularizing, amplifying, and clusteringthen enables the use of sequencing technologies, such as avidity basedsequencing methods such as those described in U.S. Application Nos.62/897,172 and Ser. No. 16/579,794, which are hereby incorporated byreference in their entireties; and as described elsewhere herein.Enablement of cellularly or tissue addressable sequencing is furtherprovided by advances in low-binding surfaces, as disclosed in U.S.patent application Ser. No. 16/363,842, hybridization methods asdisclosed in U.S. patent application Ser. No. 16/543,351, and librarypreparation methods as disclosed in U.S. Application Nos. 62/767,943 andrelated published International Application No. WO 2020/102766, thecontents of which are hereby expressly incorporated by reference for allpurposes. Thus, in some embodiments, sequence data can be obtained in amanner that maps spatially to the cell or tissue from which the genomicor transcriptomic nucleic acids were obtained. In some embodiments, thesequence data can be obtained with a substantially one-to-onecorrespondence with the cellular location of the origin of the sample.In some embodiments, the sequence data can be obtained with other than aone-to-one spatial correspondence with the cellular locations within theoriginal sample, but with substantially the same locations relative toother cells or sources of genetic, genomic, or transcriptomic sampleswithin the tissue.

Solid Support Surfaces. Provided herein solid supports comprisingsurfaces (e.g., low non-specific binding). In some instances, the solidsupport comprises a surface that is not hydrophilic. In some instances,the solid support comprises a surface that is hydrophilic. In general,the disclosed supports may comprise a substrate (or support structure),one or more layers of a covalently or non-covalently attachedlow-binding, chemical modification layers, e.g., silane layers, polymerfilms, and one or more covalently or non-covalently attached primersequences that may be used for tethering single-stranded templateoligonucleotides to the support surface (FIG. 1). In some instances, theformulation of the surface, e.g., the chemical composition of one ormore layers, the coupling chemistry used to cross-link the one or morelayers to the support surface and/or to each other, and the total numberof layers, may be varied such that non-specific binding of proteins,nucleic acid molecules, and other hybridization and amplificationreaction components to the support surface is minimized or reducedrelative to a comparable monolayer. Often, the formulation of thesurface may be varied such that non-specific hybridization on thesupport surface is minimized or reduced relative to a comparablemonolayer. The formulation of the surface may be varied such thatnon-specific amplification on the support surface is minimized orreduced relative to a comparable monolayer. The formulation of thesurface may be varied such that specific amplification rates and/oryields on the support surface are maximized. Amplification levelssuitable for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, or more than 30 amplification cycles in somecases disclosed herein.

Examples of materials from which the substrate or support structure maybe fabricated include, but are not limited to, glass, fused-silica,silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof. Various compositionsof both glass and plastic substrates are contemplated.

The substrate or support structure may be rendered in any of a varietyof geometries and dimensions known to those of skill in the art, and maycomprise any of a variety of materials known to those of skill in theart. For example, in some instances the substrate or support structuremay be locally planar (e.g., comprising a microscope slide or thesurface of a microscope slide). Globally, the substrate or supportstructure may be cylindrical (e.g., comprising a capillary or theinterior surface of a capillary), spherical (e.g., comprising the outersurface of a non-porous bead), or irregular (e.g., comprising the outersurface of an irregularly-shaped, non-porous bead or particle). In someinstances, the surface of the substrate or support structure used fornucleic acid hybridization and amplification may be a solid, non-poroussurface. In some instances, the surface of the substrate or supportstructure used for nucleic acid hybridization and amplification may beporous, such that the coatings described herein penetrate the poroussurface, and nucleic acid hybridization and amplification reactionsperformed thereon may occur within the pores.

The substrate or support structure that comprises the one or morechemically-modified layers, e.g., layers of a low non-specific bindingpolymer, may be independent or integrated into another structure orassembly. For example, in some instances, the substrate or supportstructure may comprise one or more surfaces within an integrated orassembled microfluidic flow cell. The substrate or support structure maycomprise one or more surfaces within a microplate format, e.g., thebottom surface of the wells in a microplate. As noted above, in somepreferred embodiments, the substrate or support structure comprises theinterior surface (such as the lumen surface) of a capillary. Inalternate preferred embodiments the substrate or support structurecomprises the interior surface (such as the lumen surface) of acapillary etched into a planar chip.

The chemical modification layers may be applied uniformly across thesurface of the substrate or support structure. Alternately, the surfaceof the substrate or support structure may be non-uniformly distributedor patterned, such that the chemical modification layers are confined toone or more discrete regions of the substrate. For example, thesubstrate surface may be patterned using photolithographic techniques tocreate an ordered array or random pattern of chemically-modified regionson the surface. Alternately or in combination, the substrate surface maybe patterned using, e.g., contact printing and/or ink-jet printingtechniques. In some instances, an ordered array or random pattern ofchemically-modified discrete regions may comprise at least 1, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or morediscrete regions, or any intermediate number spanned by the rangeherein.

In order to achieve low non-specific binding surfaces (also referred toherein as “low binding” or “passivated” surfaces), hydrophilic polymersmay be non-specifically adsorbed or covalently grafted to the substrateor support surface. Typically, passivation is performed utilizingpoly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) orpolyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilicpolymers with different molecular weights and end groups that are linkedto a surface using, for example, silane chemistry. The end groups distalfrom the surface can include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In someinstances, two or more layers of a hydrophilic polymer, e.g., a linearpolymer, branched polymer, or multi-branched polymer, may be depositedon the surface. In some instances, two or more layers may be covalentlycoupled to each other or internally cross-linked to improve thestability of the resulting surface. In some instances, oligonucleotideprimers with different base sequences and base modifications (or otherbiomolecules, e.g., enzymes or antibodies) may be tethered to theresulting surface layer at various surface densities. In some instances,for example, both surface functional group density and oligonucleotideconcentration may be varied to target a certain primer density range.Additionally, primer density can be controlled by dilutingoligonucleotide with other molecules that carry the same functionalgroup. For example, amine-labeled oligonucleotide can be diluted withamine-labeled polyethylene glycol in a reaction with an NETS-estercoated surface to reduce the final primer density. Primers withdifferent lengths of linker between the hybridization region and thesurface attachment functional group can also be applied to controlsurface density. Example of suitable linkers include poly-T and poly-Astrands at the 5′ end of the primer (e.g., 0 to 20 bases), PEG linkers(e.g., 3 to 20 monomer units), and carbon-chain (e.g., C6, C12, C18,etc.). To measure the primer density, fluorescently-labeled primers maybe tethered to the surface and a fluorescence reading then compared withthat for a dye solution of known concentration.

In some embodiments, the hydrophilic polymer can be a cross linkedpolymer. In some embodiments, the cross-linked polymer can include onetype of polymer cross linked with another type of polymer. Examples ofthe crossed-linked polymer can include poly(ethylene glycol)cross-linked with another polymer selected from polyethylene oxide (PEO)or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilicpolymers. In some embodiments, the cross-linked polymer can be apoly(ethylene glycol) cross-linked with polyacrylamide.

As a result of the surface passivation techniques disclosed herein,proteins, nucleic acids, and other biomolecules do not “stick” to thesubstrates, that is, they exhibit low nonspecific binding (NSB).Examples are shown below using standard monolayer surface preparationswith varying glass preparation conditions. Hydrophilic surface that havebeen passivated to achieve ultra-low NSB for proteins and nucleic acidsrequire novel reaction conditions to improve primer deposition reactionefficiencies, hybridization performance, and induce effectiveamplification. All of these processes require oligonucleotide attachmentand subsequent protein binding and delivery to a low binding surface. Asdescribed below, the combination of a new primer surface conjugationformulation (Cy3 oligonucleotide graft titration) and resultingultra-low non-specific background (NSB functional tests performed usingred and green fluorescent dyes) yielded results that demonstrate theviability of the disclosed approaches. Some surfaces disclosed hereinexhibit a ratio of specific (e.g., hybridization to a tethered primer orprobe) to nonspecific binding (e.g., B_(inter)) of a fluorophore such asCy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediatevalue spanned by the range herein. Some surfaces disclosed hereinexhibit a ratio of specific to nonspecific fluorescence signal (e.g.,for specifically-hybridized to nonspecifically bound labeledoligonucleotides, or for specifically-amplified to nonspecifically-bound(B_(inter)) or non-specifically amplified (B_(intra)) labeledoligonucleotides or a combination thereof (B_(inter)+B_(intra))) for afluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or anyintermediate value spanned by the range herein.

In order to scale primer surface density and add additionaldimensionality to hydrophilic or amphoteric surfaces, substratescomprising multi-layer coatings of PEG and other hydrophilic polymershave been developed. By using hydrophilic and amphoteric surfacelayering approaches that include, but are not limited to, thepolymer/co-polymer materials described below, it is possible to increaseprimer loading density on the surface significantly. Traditional PEGcoating approaches use monolayer primer deposition, which have beengenerally reported for single molecule applications, but do not yieldhigh copy numbers for nucleic acid amplification applications. Asdescribed herein “layering” can be accomplished using traditionalcrosslinking approaches with any compatible polymer or monomer subunitssuch that a surface comprising two or more highly crosslinked layers canbe built sequentially. Examples of suitable polymers include, but arenot limited to, streptavidin, poly acrylamide, polyester, dextran,poly-lysine, and copolymers of poly-lysine and PEG. In some instances,the different layers may be attached to each other through any of avariety of conjugation reactions including, but not limited to,biotin-streptavidin binding, azide-alkyne click reaction, amine-NETSester reaction, thiol-maleimide reaction, and ionic interactions betweenpositively charged polymer and negatively charged polymer. In someinstances, high primer density materials may be constructed in solutionand subsequently layered onto the surface in multiple steps.

The attachment chemistry used to graft a first chemically-modified layerto a support surface will generally be dependent on both the materialfrom which the support is fabricated and the chemical nature of thelayer. In some instances, the first layer may be covalently attached tothe support surface. In some instances, the first layer may benon-covalently attached, e.g., adsorbed to the surface throughnon-covalent interactions such as electrostatic interactions, hydrogenbonding, or van der Waals interactions between the surface and themolecular components of the first layer. In either case, the substratesurface may be treated prior to attachment or deposition of the firstlayer. Any of a variety of surface preparation techniques known to thoseof skill in the art may be used to clean or treat the support surface.For example, glass or silicon surfaces may be acid-washed using aPiranha solution (a mixture of sulfuric acid (H2SO4) and hydrogenperoxide (H2O2)) and/or cleaned using an oxygen plasma treatment method.

Silane chemistries constitute one non-limiting approach for covalentlymodifying the silanol groups on glass or silicon surfaces to attach morereactive functional groups (e.g., amines or carboxyl groups), which maythen be used in coupling linker molecules (e.g., linear hydrocarbonmolecules of various lengths, such as C6, C12, C18 hydrocarbons, orlinear polyethylene glycol (PEG) molecules) or layer molecules (e.g.,branched PEG molecules or other polymers) to the surface. Examples ofsuitable silanes that may be used in creating any of the disclosed lowbinding support surfaces include, but are not limited to,(3-Aminopropyl) trimethoxysilane (APTMS), (3-Aminopropyl)triethoxysilane (APTES), any of a variety of PEG-silanes (e.g.,comprising molecular weights of 1 K, 2 K, 5 K, 10 K, 20 K, etc.),amino-PEG silane (i.e., comprising a free amino functional group),maleimide-PEG silane, biotin-PEG silane, and the like.

Any of a variety of molecules known to those of skill in the artincluding, but not limited to, amino acids, peptides, nucleotides,oligonucleotides, other monomers or polymers, or combinations thereofmay be used in creating the one or more chemically-modified layers onthe support surface, where the choice of components used may be variedto alter one or more properties of the support surface, e.g., thesurface density of functional groups and/or tethered oligonucleotideprimers, the hydrophilicity/hydrophobicity of the support surface, orthe three three-dimensional nature (i.e., “thickness”) of the supportsurface. Examples of preferred polymers that may be used to create oneor more layers of low non-specific binding material in any of thedisclosed support surfaces include, but are not limited to, polyethyleneglycol (PEG) of various molecular weights and branching structures,streptavidin, polyacrylamide, polyester, dextran, poly-lysine, andpoly-lysine copolymers, or any combination thereof. Examples ofconjugation chemistries that may be used to graft one or more layers ofmaterial (e.g. polymer layers) to the support surface and/or tocross-link the layers to each other include, but are not limited to,biotin-streptavidin interactions (or variations thereof), his tag—Ni/NTAconjugation chemistries, methoxy ether conjugation chemistries,carboxylate conjugation chemistries, amine conjugation chemistries, NHSesters, maleimides, thiol, epoxy, azide, hydrazide, alkyne, isocyanate,and silane.

One or more layers of a multi-layered surface may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(2-hydroxylethylmethacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol)methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid(branched PGA), branched poly-lysine, branched poly-glucoside, anddextran.

In some instances, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branches. Molecules often exhibit a ‘power of 2’ number of branches,such as 2, 4, 8, 16, 32, 64, or 128 branches.

Exemplary PEG multilayers include PEG (8, 16, 8) (8 arm, 16 arm, 8 arm)?on PEG-amine-APTES. Similar concentrations were observed for 3-layermulti-arm PEG (8 arm, 16 arm, 8 arm) and (8 arm, 64 arm, 8 arm) onPEG-amine-APTES exposed to 8 uM primer, and 3-layer multi-arm PEG (8arm, 8 arm, 8 arm) using star-shape PEG-amine to replace 16 arm and 64arm PEG multilayers having comparable first, second and third PEG layersare also contemplated.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500, at least 1,000, at least 1,500, atleast 2,000, at least 2,500, at least 3,000, at least 3,500, at least4,000, at least 4,500, at least 5,000, at least 7,500, at least 10,000,at least 12,500, at least 15,000, at least 17,500, at least 20,000, atleast 25,000, at least 30,000, at least 35,000, at least 40,000, atleast 45,000, or at least 50,000 Daltons. In some instances, the linear,branched, or multi-branched polymers used to create one or more layersof any of the multi-layered surfaces disclosed herein may have amolecular weight of at most 50,000, at most 45,000, at most 40,000, atmost 35,000, at most 30,000, at most 25,000, at most 20,000, at most17,500, at most 15,000, at most 12,500, at most 10,000, at most 7,500,at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, or atmost 500 Daltons. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the molecular weight oflinear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may rangefrom about 1,500 to about 20,000 Daltons. Those of skill in the art willrecognize that the molecular weight of linear, branched, ormulti-branched polymers used to create one or more layers of any of themulti-layered surfaces disclosed herein may have any value within thisrange, e.g., about 1,260 Daltons.

In some instances, e.g., wherein at least one layer of a multi-layeredsurface comprises a branched polymer, the number of covalent bondsbetween a branched polymer molecule of the layer being deposited andmolecules of the previous layer may range from about one covalentlinkage per molecule and about 32 covalent linkages per molecule. Insome instances, the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the previous layer may be atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 12, at least 14,at least 16, at least 18, at least 20, at least 22, at least 24, atleast 26, at least 28, at least 30, or at least 32, or more than 32covalent linkages per molecule. In some instances, the number ofcovalent bonds between a branched polymer molecule of the new layer andmolecules of the previous layer may be at most 32, at most 30, at most28, at most 26, at most 24, at most 22, at most 20, at most 18, at most16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7,at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the number of covalent bonds between abranched polymer molecule of the new layer and molecules of the previouslayer may range from about 4 to about 16. Those of skill in the art willrecognize that the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the previous layer may haveany value within this range, e.g., about 11 in some instances, or anaverage number of about 4.6 in other instances.

Any reactive functional groups that remain following the coupling of amaterial layer to the support surface may optionally be blocked bycoupling a small, inert molecule using a high yield coupling chemistry.For example, in the case that amine coupling chemistry is used to attacha new material layer to the previous one, any residual amine groups maysubsequently be acetylated or deactivated by coupling with a small aminoacid such as glycine.

The number of layers of low non-specific binding material, e.g., ahydrophilic polymer material, deposited on the surface of the disclosedlow binding supports may range from 1 to about 10. In some instances,the number of layers is at least 1, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10. In some instances, the number of layers may be at most 10, at most9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, atmost 2, or at most 1. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the number of layersmay range from about 2 to about 4. In some instances, all of the layersmay comprise the same material. In some instances, each layer maycomprise a different material. In some instances, the plurality oflayers may comprise a plurality of materials. In some instances at leastone layer may comprise a branched polymer. In some instance, all of thelayers may comprise a branched polymer.

One or more layers of low non-specific binding material may in somecases be deposited on and/or conjugated to the substrate surface using apolar protic solvent, a polar aprotic solvent, a nonpolar solvent, orany combination thereof. In some instances the solvent used for layerdeposition and/or coupling may comprise an alcohol (e.g., methanol,ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile,dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), etc.), water, anaqueous buffer solution (e.g., phosphate buffer, phosphate bufferedsaline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or anycombination thereof. In some instances, an organic component of thesolvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% of the total, or any percentage spanned or adjacent to the rangeherein, with the balance made up of water or an aqueous buffer solution.In some instances, an aqueous component of the solvent mixture used maycomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or anypercentage spanned or adjacent to the range herein, with the balancemade up of an organic solvent. The pH of the solvent mixture used may beless than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greaterthan 10, or any value spanned or adjacent to the range described herein.

In some instances, one or more layers of low non-specific bindingmaterial may be deposited on and/or conjugated to the substrate surfaceusing a mixture of organic solvents, wherein the dielectric constant ofat least once component is less than 40 and constitutes at least 50% ofthe total mixture by volume. In some instances, the dielectric constantof the at least one component may be less than 10, less than 20, lessthan 30, less than 40. In some instances, the at least one componentconstitutes at least 20%, at least 30%, at least 40%, at least 50%, atleast 50%, at least 60%, at least 70%, or at least 80% of the totalmixture by volume.

As noted, the low non-specific binding supports of the presentdisclosure exhibit reduced non-specific binding of proteins, nucleicacids, and other components of the hybridization and/or amplificationformulation used for solid-phase nucleic acid amplification. The degreeof non-specific binding exhibited by a given support surface may beassessed either qualitatively or quantitatively. For example, in someinstances, exposure of the surface to fluorescent dyes (e.g., Cy3, Cy5,etc.), fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, and/or fluorescently-labeled proteins (e.g.polymerases) under a standardized set of conditions, followed by aspecified rinse protocol and fluorescence imaging may be used as aqualitative tool for comparison of non-specific binding on supportscomprising different surface formulations. In some instances, exposureof the surface to fluorescent dyes, fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, and/or fluorescently-labeledproteins (e.g. polymerases) under a standardized set of conditions,followed by a specified rinse protocol and fluorescence imaging may beused as a quantitative tool for comparison of non-specific binding onsupports comprising different surface formulations—provided that carehas been taken to ensure that the fluorescence imaging is performedunder conditions where fluorescence signal is linearly related (orrelated in a predictable manner) to the number of fluorophores on thesupport surface (e.g., under conditions where signal saturation and/orself-quenching of the fluorophore is not an issue) and suitablecalibration standards are used. In some instances, other techniquesknown to those of skill in the art, for example, radioisotope labelingand counting methods may be used for quantitative assessment of thedegree to which non-specific binding is exhibited by the differentsupport surface formulations of the present disclosure.

Some surfaces disclosed herein exhibit a ratio of specific tonon-specific binding of a fluorophore such as Cy3 of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 50, 75, 100, or greater than 100, or any intermediate value spannedby the range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to non-specific fluorescence of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein.

As noted, in some instances, the degree of non-specific bindingexhibited by the disclosed low non-specific binding supports may beassessed using a standardized protocol for contacting the surface with alabeled protein (e.g., bovine serum albumin (BSA), streptavidin, a DNApolymerase, a reverse transcriptase, a helicase, a single-strandedbinding protein (SSB), etc., or any combination thereof), a labelednucleotide, a labeled oligonucleotide, etc., under a standardized set ofincubation and rinse conditions, followed be detection of the amount oflabel remaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label known to one of skill in the art. In someinstances, the degree of non-specific binding exhibited by a givensupport surface formulation may thus be assessed in terms of the numberof non-specifically bound protein molecules (or other molecules) perunit area. In some instances, the low non-specific binding supports ofthe present disclosure may exhibit non-specific protein binding (ornon-specific binding of other specified molecules, e.g., Cy3 dye) ofless than 0.001 molecule per μm2, less than 0.01 molecule per μm2, lessthan 0.1 molecule per μm2, less than 0.25 molecule per μm2, less than0.5 molecule per μm2, less than 1 molecule per μm2, less than 10molecules per μm2, less than 100 molecules per μm2, or less than 1,000molecules per μm2. Those of skill in the art will realize that a givensupport surface of the present disclosure may exhibit non-specificbinding falling anywhere within this range, for example, of less than 86molecules per μm2. For example, some modified surfaces disclosed hereinexhibit non-specific protein binding of less than 0.5 molecule/μm2following contact with a 1 uM solution of Cy3 labeled streptavidin (GEAmersham) in phosphate buffered saline (PBS) buffer for 15 minutes,followed by 3 rinses with deionized water. Some modified surfacesdisclosed herein exhibit non-specific binding of Cy3 dye molecules ofless than 0.25 molecules per um2. In independent non-specific bindingassays, 1 uM labeled Cy3 SA (ThermoFisher), 1 uM Cy5 SA dye(ThermoFisher), 10 uM Aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10uM Aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 uMAminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 uM7-Propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences, and 10 uM7-Propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated onthe low binding substrates at 37° C. for 15 minutes in a 384 well plateformat. Each well was rinsed 2-3× with 50 ul deionized RNase/DNase Freewater and 2-3× with 25 mM ACES buffer pH 7.4. The 384 well plates wereimaged on a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, Pa.)instrument using the Cy3, AF555, or Cy5 filter sets (according to dyetest performed) as specified by the manufacturer at a PMT gain settingof 800 and resolution of 50-100 μm. For higher resolution imaging,images were collected on an Olympus IX83 microscope (Olympus Corp.,Center Valley, Pa.) with a total internal reflectance fluorescence(TIRF) objective (20×, 0.75 NA or 100×, 1.5 NA, Olympus), an sCMOS Andorcamera (Zyla 4.2), and excitation wavelengths of 532 nm or 635 nm.Dichroic mirrors were purchased from Semrock (IDEX Health & Science,LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633 nm dichroicreflectors/beamsplitters, and band pass filters were chosen as 532 LP or645 LP concordant with the appropriate excitation wavelength. Somemodified surfaces disclosed herein exhibit non-specific binding of dyemolecules of less than 0.25 molecules per μm2.

In some instances, the surfaces disclosed herein exhibit a ratio ofspecific to non-specific binding of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein. In some instances, thesurfaces disclosed herein exhibit a ratio of specific to non-specificfluorescence signals for a fluorophore such as Cy3 of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 50, 75, 100, or greater than 100, or any intermediate value spannedby the range herein.

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or morethan 50 specific dye molecules attached per molecule non-specificallyadsorbed. Similarly, when subjected to an excitation energy,low-background surfaces consistent with the disclosure herein to whichfluorophores, e.g., Cy3, have been attached may exhibit ratios ofspecific fluorescence signal (e.g., arising from Cy3-labeledoligonucleotides attached to the surface) to non-specific adsorbed dyefluorescence signals of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces may be assessed,for example, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome instances, a static contact angle may be determined. In someinstances, an advancing or receding contact angle may be determined. Insome instances, the water contact angle for the hydrophilic, low-bindingsupport surfaced disclosed herein may range from about 0 degrees toabout 50 degrees. In some instances, the water contact angle for thehydrophilic, low-binding support surfaced disclosed herein may no morethan 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. Inmany cases the contact angle is no more than any value within thisrange, e.g., no more than 40 degrees. Those of skill in the art willrealize that a given hydrophilic, low-binding support surface of thepresent disclosure may exhibit a water contact angle having a value ofanywhere within this range, e.g., about 27 degrees.

In some instances, the hydrophilic surfaces disclosed herein facilitatereduced wash times for bioassays, often due to reduced non-specificbinding of biomolecules to the low-binding surfaces. In some instances,adequate wash steps may be performed in less than 60, 50, 40, 30, 20,15, 10, or less than 10 seconds. For example, in some instances adequatewash steps may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantimprovement in stability or durability to prolonged exposure to solventsand elevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. For example, in some instances, the stability ofthe disclosed surfaces may be tested by fluorescently labeling afunctional group on the surface, or a tethered biomolecule (e.g., anoligonucleotide primer) on the surface, and monitoring fluorescencesignal before, during, and after prolonged exposure to solvents andelevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. In some instances, the degree of change in thefluorescence used to assess the quality of the surface may be less than1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a time period of 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50hours, or 100 hours of exposure to solvents and/or elevated temperatures(or any combination of these percentages as measured over these timeperiods). In some instances, the degree of change in the fluorescenceused to assess the quality of the surface may be less than 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeatedexposure to solvent changes and/or changes in temperature (or anycombination of these percentages as measured over this range of cycles).

In some instances, the surfaces disclosed herein may exhibit a highratio of specific signal to non-specific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greater than asignal of an adjacent unpopulated region of the surface. Similarly, somesurfaces exhibit an amplification signal that is at least 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greaterthan a signal of an adjacent amplified nucleic acid population region ofthe surface.

Fluorescence excitation energies vary among particular fluorophores andprotocols, and may range in excitation wavelength from less than 400 nmto over 800 nm, consistent with fluorophore selection or otherparameters of use of a surface disclosed herein.

Accordingly, low non-specific binding surfaces as disclosed hereinexhibit low background fluorescence signals or high contrast to noise(CNR) ratios relative to known surfaces in the art. For example, in someinstances, the background fluorescence of the surface at a location thatis spatially distinct or removed from a labeled feature on the surface(e.g., a labeled spot, cluster, discrete region, sub-section, or subsetof the surface) comprising a hybridized cluster of nucleic acidmolecules, or a clonally-amplified cluster of nucleic acid moleculesproduced by, e.g., 20 cycles of nucleic acid amplification viathermocycling, may be no more than 20×, 10×, 5×, 2×, 1×, 0.5×, 0.1×, orless than 0.1× greater than the background fluorescence measured at thatsame location prior to performing said hybridization or said 20 cyclesof nucleic acid amplification.

In some instances, fluorescence images of the disclosed low backgroundsurfaces when used in nucleic acid hybridization or amplificationapplications to create clusters of hybridized or clonally-amplifiednucleic acid molecules (e.g., that have been directly or indirectlylabeled with a fluorophore) exhibit contrast-to-noise ratios (CNRs) ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than250.

In general, at least one layer of the one or more layers of lownon-specific binding material may comprise functional groups forcovalently or non-covalently attaching oligonucleotide molecules, e.g.,adapter or primer sequences, or the at least one layer may alreadycomprise covalently or non-covalently attached oligonucleotide adapteror primer sequences at the time that it is deposited on the supportsurface. In some instances, the oligonucleotides tethered to the polymermolecules of at least one third layer may be distributed at a pluralityof depths throughout the layer.

In some instances, the oligonucleotide adapter or primer molecules arecovalently coupled to the polymer in solution, e.g., prior to couplingor depositing the polymer on the surface. In some instances, theoligonucleotide adapter or primer molecules are covalently coupled tothe polymer after it has been coupled to or deposited on the surface. Insome instances, at least one hydrophilic polymer layer comprises aplurality of covalently-attached oligonucleotide adapter or primermolecules. In some instances, at least two, at least three, at leastfour, or at least five layers of hydrophilic polymer comprise aplurality of covalently-attached adapter or primer molecules.

In some instances, the oligonucleotide adapter or primer molecules maybe coupled to the one or more layers of hydrophilic polymer using any ofa variety of suitable conjugation chemistries known to those of skill inthe art. For example, the oligonucleotide adapter or primer sequencesmay comprise moieties that are reactive with amine groups, carboxylgroups, thiol groups, and the like. Examples of suitable amine-reactiveconjugation chemistries that may be used include, but are not limitedto, reactions involving isothiocyanate, isocyanate, acyl azide, NHSester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,carbonate, aryl halide, imidoester, carbodiimide, anhydride, andfluorophenyl ester groups. Examples of suitable carboxyl-reactiveconjugation chemistries include, but are not limited to, reactionsinvolving carbodiimide compounds, e.g., water soluble EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCL). Examples ofsuitable sulfydryl-reactive conjugation chemistries include maleimides,haloacetyls and pyridyl disulfides.

One or more types of oligonucleotide molecules may be attached ortethered to the support surface. In some instances, the one or moretypes of oligonucleotide adapters or primers may comprise spacersequences, adapter sequences for hybridization to adapter-ligatedtemplate library nucleic acid sequences, forward amplification primers,reverse amplification primers, sequencing primers, and/or molecularbarcoding sequences, or any combination thereof. In some instances, 1primer or adapter sequence may be tethered to at least one layer of thesurface. In some instances, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 different primer or adapter sequences may be tethered to atleast one layer of the surface.

The tethered oligonucleotide adapter and/or primer sequences may rangein length from about 10 nucleotides to about 100 nucleotides. In someinstances, the tethered oligonucleotide adapter and/or primer sequencesmay be at least 10, at least 20, at least 30, at least 40, at least 50,at least 60, at least 70, at least 80, at least 90, or at least 100nucleotides in length. In some instances, the tethered oligonucleotideadapter and/or primer sequences may be at most 100, at most 90, at most80, at most 70, at most 60, at most 50, at most 40, at most 30, at most20, or at most 10 nucleotides in length. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe length of the tethered oligonucleotide adapter and/or primersequences may range from about 20 nucleotides to about 80 nucleotides.Those of skill in the art will recognize that the length of the tetheredoligonucleotide adapter and/or primer sequences may have any valuewithin this range, e.g., about 24 nucleotides.

In some instances, the tethered adapter or primer sequences may comprisemodifications designed to facilitate the specificity and efficiency ofnucleic acid amplification as performed on the low-binding supports. Forexample, in some instances the primer may comprise polymerase stoppoints such that the stretch of primer sequence between the surfaceconjugation point and the modification site is always in single-strandedform and functions as a loading site for 5′ to 3′ helicases in somehelicase-dependent isothermal amplification methods. Other examples ofprimer modifications that may be used to create polymerase stop pointsinclude, but are not limited to, an insertion of a PEG chain into thebackbone of the primer between two nucleotides towards the 5′ end,insertion of an abasic nucleotide (i.e., a nucleotide that has neither apurine nor a pyrimidine base), or a lesion site which can be bypassed bythe helicase.

As will be discussed further in the examples below, it may be desirableto vary the surface density of tethered oligonucleotide adapters orprimers on the support surface and/or the spacing of the tetheredadapters or primers away from the support surface (e.g., by varying thelength of a linker molecule used to tether the adaptors or primers tothe surface) in order to “tune” the support for optimal performance whenusing a given amplification method. As noted below, adjusting thesurface density of tethered oligonucleotide adapters or primers mayimpact the level of specific and/or non-specific amplification observedon the support in a manner that varies according to the amplificationmethod selected. In some instances, the surface density of tetheredoligonucleotide adapters or primers may be varied by adjusting the ratioof molecular components used to create the support surface. For example,in the case that an oligonucleotide primer—PEG conjugate is used tocreate the final layer of a low-binding support, the ratio of theoligonucleotide primer—PEG conjugate to a non-conjugated PEG moleculemay be varied. The resulting surface density of tethered primermolecules may then be estimated or measured using any of a variety oftechniques known to those of skill in the art. Examples include, but arenot limited to, the use of radioisotope labeling and counting methods,covalent coupling of a cleavable molecule that comprises anoptically-detectable tag (e.g., a fluorescent tag) that may be cleavedfrom a support surface of defined area, collected in a fixed volume ofan appropriate solvent, and then quantified by comparison offluorescence signals to that for a calibration solution of known opticaltag concentration, or using fluorescence imaging techniques providedthat care has been taken with the labeling reaction conditions and imageacquisition settings to ensure that the fluorescence signals arelinearly related to the number of fluorophores on the surface (e.g.,that there is no significant self-quenching of the fluorophores on thesurface).

In some instances, the resultant surface density of oligonucleotideadapters or primers on the low binding support surfaces of the presentdisclosure may range from about 100 primer molecules per μm2 to about1,000,000 primer molecules per μm2. In some instances, the surfacedensity of oligonucleotide adapters or primers may be at least 100, atleast 200, at least 300, at least 400, at least 500, at least 600, atleast 700, at least 800, at least 900, at least 1,000, at least 1,500,at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000,at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000,at least 20,000, at least 25,000, at least 30,000, at least 35,000, atleast 40,000, at least 45,000, at least 50,000, at least 55,000, atleast 60,000, at least 65,000, at least 70,000, at least 75,000, atleast 80,000, at least 85,000, at least 90,000, at least 95,000, atleast 100,000, at least 150,000, at least 200,000, at least 250,000, atleast 300,000, at least 350,000, at least 400,000, at least 450,000, atleast 500,000, at least 550,000, at least 600,000, at least 650,000, atleast 700,000, at least 750,000, at least 800,000, at least 850,000, atleast 900,000, at least 950,000, or at least 1,000,000 molecules perμm2. In some instances, the surface density of oligonucleotide adaptersor primers may be at most 1,000,000, at most 950,000, at most 900,000,at most 850,000, at most 800,000, at most 750,000, at most 700,000, atmost 650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, atmost 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000,at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most800, at most 700, at most 600, at most 500, at most 400, at most 300, atmost 200, or at most 100 molecules per μm2. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe surface density of adapters or primers may range from about 10,000molecules per μm2 to about 100,000 molecules per μm2. Those of skill inthe art will recognize that the surface density of adapter or primermolecules may have any value within this range, e.g., about 3,800molecules per μm2 in some instances, or about 455,000 molecules per μm2in other instances. In some instances, as will be discussed furtherbelow, the surface density of template library nucleic acid sequences(e.g., sample DNA molecules) initially hybridized to adapter or primersequences on the support surface may be less than or equal to thatindicated for the surface density of tethered oligonucleotide primers.In some instances, as will also be discussed further below, the surfacedensity of clonally-amplified template library nucleic acid sequenceshybridized to adapter or primer sequences on the support surface mayspan the same range or a different range as that indicated for thesurface density of tethered oligonucleotide adapters or primers.

Local surface densities of adapter or primer molecules as listed abovedo not preclude variation in density across a surface, such that asurface may comprise a region having an oligo density of, for example,500,000/μm2, while also comprising at least a second region having asubstantially different local density.

Solid Supports for Capturing and Analyzing DNA. In some embodiments, thesurface has bound thereto a plurality of oligonucleotides for thecapture of target nucleic acids, such as DNA molecules (e.g., captureoligonucleotides; (1)), as shown in FIG. 2. In some embodiments, thecapture oligonucleotides each comprise single-stranded oligonucleotides.The capture oligonucleotides can be immobilized to the passivatedsurface by their 5′ ends, or an internal portion of the captureoligonucleotides can be immobilized to the passivated surface. Thecapture oligonucleotides can each include an extendible 3′ end. As shownin FIG. 2, the capture oligonucleotides can each include a cleavableregion (6) which can be located near the end that is immobilized to thepassivated surface. For example, the capture oligonucleotides can eachinclude a cleavable region near the 5′ end. The cleavable region can becleaved with an enzyme, a chemical compound, light or heat. In someembodiments, the capture oligonucleotides each comprise a target captureregion (2) and a universal sequence region (3, 4, 5). In someembodiments, the target capture region of the capture oligonucleotidescomprise a sequence that can hybridize to at least a portion of thetarget nucleic acid. The target capture region may comprise, forexample, a random nucleotide sequence or a target-specific sequence thatcorresponds to a known sequence of the target nucleic acid. In someembodiments, the universal sequence region comprises a sample barcodesequence (3) that can be used to distinguish target nucleic acids fromdifferent sample sources in a multiplex assay. In some embodiments, theuniversal sequence region comprises a spatial barcode sequence (4) whichconveys positional information of the capture oligonucleotide on thesupport which in turn conveys positional information of the cell withinthe tissue sample or of a single cell. In some embodiments, the samplebarcode sequence (3) can be upstream or downstream of the spatialbarcode sequence (4). In some embodiments, the universal sequence regionof the capture oligonucleotides comprise a circularization anchor region(5) that hybridizes to a portion of a second type of oligonucleotidethat promotes circularization of the captured nucleic acid (7). In someembodiments, the universal sequence region of the captureoligonucleotides comprise at least one sequence that binds/hybridizes toa universal primer sequence such as a sequencing primer sequence and/oran amplification primer sequence. In some embodiments, thecircularization anchor region (5) includes any one or any combination oftwo or more of the sequencing primer sequence, the amplification primersequence, the sample barcode sequence and/or the spatial barcodesequence. In some embodiments, the circularization anchor region (5)comprises a separate sequence that hybridizes with a portion of thesecond type of oligonucleotide that promotes circularization of thecaptured nucleic acid. In some embodiments, the universal sequenceregion comprises a cleavable region which is cleavable with an enzyme, achemical compound, light or heat.

Still referring to FIG. 2, in some embodiments, the surface has boundthereto a plurality of a second type of oligonucleotide (e.g.,circularization oligonucleotides (7)) that promote circularization ofthe captured target nucleic acids. In some embodiments, thecircularization oligonucleotides each comprise single-strandedoligonucleotides. The circularization oligonucleotides can beimmobilized to the passivated surface by their 5′ ends, or an internalportion of the circularization oligonucleotides can be immobilized tothe passivated surface. The circularization oligonucleotides can eachinclude an extendible 3′ end. The circularization oligonucleotides eachcomprise a homopolymer region (8) and a universal sequence region (9),as shown in FIG. 2. The homopolymer region can be selected from a groupconsisting of poly-T tail, poly-dT tail, poly-A tail, poly-dA tail,poly-C tail, poly-dC tail, poly-G tail and poly-dG tail. The homopolymerregion can be located at or near the 3′ end of the circularizationoligonucleotides. In some embodiments, the universal sequence region ofthe circularization oligonucleotides hybridizes to the circularizationanchor region of the capture oligonucleotides. In some embodiments, theuniversal sequence region of the circularization oligonucleotidescomprise at least one sequence that binds/hybridizes to a universalprimer sequence such as a sequencing primer sequence of the captureoligonucleotides. In some embodiments, the universal sequence region ofthe circularization oligonucleotides comprise at least one sequence thatbinds/hybridizes to a universal primer sequence such as an amplificationprimer sequence of the capture oligonucleotides. In some embodiments,the universal sequence region of the circularization oligonucleotidescomprise at least one sequence that binds/hybridizes to the samplebarcode sequence and/or the spatial barcode sequence of the captureoligonucleotides. In some embodiments, the circularizationoligonucleotides comprise a separate sequence that binds/hybridizes witha portion of the circularization anchor region of the captureoligonucleotides (e.g., a circularization anchor binding sequence).

In some embodiments, the capture oligonucleotides (FIG. 2, 1) and thecircularization oligonucleotides (FIG. 2, 7) can be immobilized on thepassivated surface prior to contacting the passivated surface with thetarget nucleic acid molecules for the target molecule capturing steps.In an alternative embodiment, the capture oligonucleotides isimmobilized on the passivated surface prior to contacting the passivatedsurface with the target nucleic acid molecules for the target moleculecapturing steps, and subsequently the plurality of circularizationoligonucleotides (e.g., in soluble form) can be provided in solution andflowed onto the passivated surface to immobilize the circularizationoligonucleotides.

In some embodiments, said circularization oligo may be the same as, maycomprise, or may be comprised within, said capture oligo. In someembodiments, said circularization oligo may comprise a separatemolecule.

The present disclosure provides a low-binding support having a coatingwhere the coating provides a low non-specific binding surface toproteins, carbohydrates, lipids, cell debris, or solution borne dyemolecules. In some embodiments, a tissue sample or cells or a singlecell can be place on the surface of the support (FIG. 3, left). In someembodiments, the low non-specific binding surface comprises a pluralityof regions (e.g., features) located at different pre-determinedlocations on the support (FIG. 3, right). The different features on thesupport can be placed at non-overlapping positions or at overlappingpositions on the support. The features can be configured to have anyshape, for example circular, ovular, square, rectangular, or polygonal.The features can be arranged in a grid pattern having rows and columns,or can be arranged in a row or a column. In some embodiments, any givenfeature contains a plurality of capture oligonucleotides and a pluralityof circularization oligonucleotides immobilized to the coating. Theplurality of features includes at least a first and second feature.

In some embodiments, the first feature comprises a plurality of firstcapture oligonucleotides having a first target capture region, a firstspatial barcode sequence, a first sample barcode sequence and a firstcleavable region, and the first feature comprises a plurality of firstcircularization oligonucleotides having a first circularization anchorbinding sequence, a first amplification primer binding sequence and afirst sequencing primer binding sequence. In some embodiments, the firstcapture oligonucleotides also include a first amplification primerbinding sequence and/or a first amplification primer binding sequence.In some embodiments, the first circularization oligonucleotides alsoinclude a sequence that can bind/hybridize to the first spatial barcodesequence and/or a sequence that can bind to the first sample barcodesequence.

In some embodiments, the second feature comprises a plurality of secondcapture oligonucleotides having a second target capture region, a secondspatial barcode sequence, a second sample barcode sequence and a secondcleavable region, and the second feature comprises a plurality of secondcircularization oligonucleotides having a second circularization anchorbinding sequence, a second amplification primer binding sequence and asecond sequencing primer binding sequence. In some embodiments, thesecond capture oligonucleotides also include a second amplificationprimer binding sequence and/or a second amplification primer bindingsequence. In some embodiments, the second circularizationoligonucleotides also include a sequence that can bind/hybridize to thesecond spatial barcode sequence and/or a sequence that can bind to thesecond sample barcode sequence.

In some embodiments, the sequence of the first target capture region inthe first feature is the same or different from the sequence of thesecond target capture region in the second feature. In some embodiments,the first spatial barcode sequence in the first feature differs from thesecond spatial barcode sequence in the second feature. In someembodiments, the first sample barcode sequence in the first feature isthe same or different as the second sample barcode sequence in thesecond feature. The first amplification primer binding sequence in thefirst feature can be the same as the second amplification primer bindingsequence in the second feature. The first sequencing primer bindingsequence in the first feature can be the same as the second sequenceprimer binding sequence in the second feature. The first cleavableregion in the first feature can be cleavable with the same or differentconditions (e.g., the same enzyme, chemical compound, light or heat) asthe second cleavable region in the second feature.

In some embodiments, the low non-specific binding coating comprises aplurality of regions (e.g., features) where the features are attachedwith a plurality of capture and circularization oligonucleotides thatare attached to the coating. In some embodiments, a first feature isattached with a first plurality of capture oligonucleotides and a firstplurality of circularization oligonucleotides, and a second feature isattached with a second plurality of capture oligonucleotides and asecond plurality of circularization oligonucleotides, wherein the firstand second capture oligonucleotides and the first and secondcircularization oligonucleotides are in fluid communication with eachother so that the capture and circularization oligonucleotides can reactwith reagents (e.g., enzymes including polymerases, polymer-nucleotideconjugates, nucleotides and/or divalent cations) in a massively parallelmanner.

In some embodiments, the cleavable region of the captureoligonucleotides are cleavable with an enzyme. In some embodiments, thecleavable region as shown in FIG. 2 (6) comprises at least one uracilbase, or a poly-uracil sequence, which is cleavable with a uracil DNAglycosylase (UDG) enzyme or a DNA glycosylase-lyase Endonuclease VIII(e.g., commercially-available enzyme USER™). In some embodiments, thecleavable site comprises at least one 8-koxoguanine (8-oxoG) which iscleavable with a DNA-formamidopyrimidine glycosylase enzyme (Fpg). Insome embodiments, the cleavable region comprises an abasic site which iscleavable with an endonuclease IV or endonuclease VIII. In someembodiments, the cleavable region which is cleavable with an enzymecomprises a nucleotide sequence which is recognized and cleaved with arestriction endonuclease enzyme which cleaves double-stranded orsingle-stranded nucleic acid strands (e.g., DNA). In some embodiments,the enzyme-cleavable region comprises a glycosidic linkage which iscleavable with an amylase enzyme, or a peptide linkage which iscleavable with a protease.

As shown in FIG. 2, in some embodiments, the cleavable region (6) of thecapture oligonucleotides is cleavable with a chemical compound comprisea labile chemical bond, for example including but not limited to esterlinkages, a thiol linkage, a vicinal diol linkage, a sulfone linkage, asilyl ether linkage, an abasic or apurinic/apyrimidinic (AP) site. Theester linkages can be cleavable with an acid, base, or hydroxylamine.The thiol linkage can be a disulfide linkage which is cleavable withglutathione or a reducing agent. The vincinal diol linkage can becleavable with sodium periodate. The sulfonate linkage can be cleavablewith a base. The silyl ether linkage can be cleavable with an acid. Theabasic or apurinic/apyrimidinic (AP) site can be cleavable with analkali or an AP endonuclease enzyme.

In some embodiments, the cleavable region (6) of the captureoligonucleotides is cleavable with light comprises a photo-cleavablemoiety which can be cleaved with exposure to light, UV light or a laser.The photo-cleavable moiety can be cleaved by exposure to any wavelengthof light. The photo-cleavable moiety comprises3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicoumarin,6-bromo-7-alkixycoumarin-4-ylmethoxycarbonyl, phenacyl esterderivatives, or 8-quinolinyl benzenesulfonate. The photo-cleavablemoiety comprises a bimane-based linker, a bis-arylhydrazone basedlinker, or an ortho-nitrobenzyl (ONB) linker. In some embodiments, thecleavable region (6) of the capture oligonucleotides is cleavable withexposure to heat comprise a Diels-Alder linker.

Supports for Capturing and Analyzing RNA. Provided herein in FIG. 4 aresupports (11) comprising a plurality of immobilized oligonucleotides.The support can be used to capture and analyze target nucleic acids, forexample RNA molecules. In some embodiments, the support comprises apassivated surface (e.g., coating or layer) (FIG. 1) which is disclosedelsewhere herein, such that the surface provides low or no binding toproteins, carbohydrates, lipids, cell debris, or solution borne dyemolecules. In some embodiments, the surface has bound thereto aplurality of oligonucleotides for the capture of target nucleic acids(e.g., capture oligonucleotides; FIG. 4 (11)). In some embodiments, thecapture oligonucleotides each comprise single-stranded oligonucleotides.The capture oligonucleotides can be immobilized to the passivatedsurface by their 5′ ends, or an internal portion of the captureoligonucleotides can be immobilized to the passivated surface. Thecapture oligonucleotides can each include an extendible 3′ end. As shownin FIG. 4, the capture oligonucleotides can each include a cleavableregion (15) which can be located near the end that is immobilized to thepassivated surface. For example, the capture oligonucleotides can eachinclude a cleavable region near the 5′ end. The cleavable region can becleaved with an enzyme, a chemical compound, light or heat. In someembodiments, the capture oligonucleotides each comprise a target captureregion (12) and a universal sequence region (13, 14). In someembodiments, the target capture region of the capture oligonucleotidescomprise a sequence that can hybridize to at least a portion of thetarget nucleic acid. The target capture region may comprise, forexample, a homopolymer sequence (e.g., poly-T or poly-dT), a randomnucleotide sequence, or a target-specific sequence that corresponds to aknown sequence of the target nucleic acid. In some embodiments, theuniversal sequence region comprises a sample barcode sequence (13) thatcan be used to distinguish target nucleic acids from different samplesources in a multiplex assay. In some embodiments, the universalsequence region comprises a spatial barcode sequence (14) which conveyspositional information of the capture oligonucleotide on the supportwhich in turn conveys positional information of the cell within thetissue sample or of a single cell. In some embodiments, the samplebarcode sequence (13) can be upstream or downstream of the spatialbarcode sequence (14). In some embodiments, the universal sequenceregion of the capture oligonucleotides comprise at least one sequencethat binds/hybridizes to a universal primer sequence such as asequencing primer sequence and/or an amplification primer sequence. Insome embodiments, the capture oligonucleotide comprises a cleavableregion (15) which is cleavable with an enzyme, a chemical compound,light or heat.

Still referring to FIG. 4, in some embodiments, provided herein are aplurality of a second type of oligonucleotide (e.g., circularizationoligonucleotides; 16) in soluble form or immobilized to the surface(e.g., coating). The circularization oligonucleotides can promotecircularization of the captured target nucleic acids. In someembodiments, the circularization oligonucleotides each comprisesingle-stranded oligonucleotides. The circularization oligonucleotidescan be in soluble form, or can be immobilized to the passivated surfaceby their 5′ ends or an internal portion of the circularizationoligonucleotides can be immobilized to the passivated surface. Thecircularization oligonucleotides can each include an extendible 3′ end.The circularization oligonucleotides each comprise an adaptor bindingregion (17). In some embodiments, the adaptor binding region includes asequencing primer binding region. In some embodiments, the adaptorbinding region include an amplification primer binding region. In someembodiments, the circularization oligonucleotides each comprise ahomopolymer region (FIG. 4 (19)). The homopolymer region can be selectedfrom a group consisting of poly-T, poly-dT, poly-A, poly-dA, poly-C,poly-dC, poly-G and poly-dG. In some embodiments, the circularizationoligonucleotides each comprise an anchor region (19) and an anchormoiety (20).

In some embodiments, the capture oligonucleotides (FIG. 4 (11)) and thecircularization oligonucleotides (FIG. 4 (16)) can be immobilized on thepassivated surface prior to contacting the passivated surface with thetarget nucleic acid molecules (e.g., RNA) for the target moleculecapturing steps. In an alternative embodiment, the captureoligonucleotides is immobilized on the passivated surface prior tocontacting the passivated surface with the target nucleic acid moleculesfor the target molecule capturing steps, and subsequently the pluralityof circularization oligonucleotides (e.g., in soluble form) can beprovided in solution and flowed onto the passivated surface toimmobilize the circularization oligonucleotides.

In some embodiments, said circularization oligo may be the same as, maycomprise, or may be comprised within, said capture oligo. In someembodiments, said circularization oligo may comprise a separatemolecule.

In some embodiments, the cleavable region (FIG. 4 (15)) of the captureoligonucleotides are cleavable with an enzyme. In some embodiments, thecleavable region comprises at least one uracil base, or a poly-uracilsequence, which is cleavable with a uracil RNA glycosylase (UDG) enzymeor a RNA glycosylase-lyase Endonuclease VIII (e.g.,commercially-available enzyme USER™). In some embodiments, the cleavablesite comprises at least one 8-koxoguanine (8-oxoG) which is cleavablewith a RNA-formamidopyrimidine glycosylase enzyme (Fpg). In someembodiments, the cleavable region comprises an abasic site which iscleavable with an endonuclease IV or endonuclease VIII. In someembodiments, the cleavable region which is cleavable with an enzymecomprises a nucleotide sequence which is recognized and cleaved with arestriction endonuclease enzyme which cleaves double-stranded orsingle-stranded nucleic acid strands (e.g., RNA). In some embodiments,the enzyme-cleavable region comprises a glycosidic linkage which iscleavable with an amylase enzyme, or a peptide linkage which iscleavable with a protease.

In some embodiments, the cleavable region (FIG. 4 (15)) of the captureoligonucleotides is cleavable with a chemical compound comprise a labilechemical bond, for example including but not limited to ester linkages,a thiol linkage, a vicinal diol linkage, a sulfone linkage, a silylether linkage, an abasic or apurinic/apyrimidinic (AP) site. The esterlinkages can be cleavable with an acid, base, or hydroxylamine. Thethiol linkage can be a disulfide linkage which is cleavable withglutathione or a reducing agent. The vincinal diol linkage can becleavable with sodium periodate. The sulfonate linkage can be cleavablewith a base. The silyl ether linkage can be cleavable with an acid. Theabasic or apurinic/apyrimidinic (AP) site can be cleavable with analkali or an AP endonuclease enzyme.

In some embodiments, the cleavable region (FIG. 4 (15)) of the captureoligonucleotides is cleavable with light comprises a photo-cleavablemoiety which can be cleaved with exposure to light, UV light or a laser.The photo-cleavable moiety can be cleaved by exposure to any wavelengthof light. The photo-cleavable moiety comprises3-amino-3-(2-nitrophenyl)propionic acid (ANP), dicoumarin,6-bromo-7-alkixycoumarin-4-ylmethoxycarbonyl, phenacyl esterderivatives, or 8-quinolinyl benzenesulfonate. The photo-cleavablemoiety comprises a bimane-based linker, a bis-arylhydrazone basedlinker, or an ortho-nitrobenzyl (ONB) linker. In some embodiments, thecleavable region (FIG. 4 (15)) of the capture oligonucleotides iscleavable with exposure to heat comprise a Diels-Alder linker.

Fixation of Biological Sample to Surfaces. Provided herein are solidsupports (e.g., low non-specific binding supports) further comprising abiological sample adjacent thereto. In some embodiments, the biologicalsample comprises a single cell, a plurality of cells, a tissue, anorgan, an organism, or section of these biological samples. In someembodiments, the biological sample is derived from eukaryotes (such asanimals, plants, fungi, protista), archaebacteria, or eubacteria. Thebiological sample may be derived from prokaryotic or eukaryotic cells,such as adherent or non-adherent eukaryotic cells. The biological samplemay be derived from a primary or immortalized cell line from a rodent,porcine, feline, canine, bovine, equine, primate, or human cell lines.

The biological sample may be a solid sample, such as a tissue biopsy.The biological sample may be a fluid sample, such as blood or acomponent of blood (e.g., serum or plasma). In some embodiments, thebiological sample is obtained from skin, heart, lung, kidney, breath,bone marrow, stool, semen, vaginal fluid, interstitial fluids derivedfrom tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue,throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle,smooth muscle, bladder, gall bladder, colon, intestine, brain, cavityfluids, sputum, pus, micropiota, meconium, breast milk, prostate,esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid,tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions,spinal fluid, hair, fingernails, skin cells, plasma, nasal swab ornasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and/orother excretions or body tissues. A biological sample may be a cell-freesample.

The biological sample may comprise cells. The cells described herein maybe white blood cells, red blood cells, platelets, epithelial cells,endothelial cells, neurons, glial cells, astrocytes, fibroblasts,skeletal muscle cells, smooth muscle cells, gametes, or cells from theheart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder,stomach, colon, or small intestine. The cells may be normal or healthycells. Alternately or in combination, the cells may be diseased cells,such as cancerous cells, or from pathogenic cells that are infecting ahost. In some embodiments, the cell belongs to a subset of cells, suchas immune cell (e.g., T cells, cytotoxic (killer) T cells, helper Tcells, alpha beta T cells, gamma delta T cells, T cell progenitors, Bcells, B-cell progenitors, lymphoid stem cells, myeloid progenitorcells, lymphocytes, granulocytes, Natural Killer cells, plasma cells,memory cells, neutrophils, eosinophils, basophils, mast cells,monocytes, dendritic cells, and/or macrophages, or any combinationthereof), undifferentiated human stem cells, human stem cells that havebeen induced to differentiate, or rare cells (e.g., circulating tumorcells (CTCs), circulating epithelial cells, circulating endothelialcells, circulating endometrial cells, bone marrow cells, progenitorcells, foam cells, mesenchymal cells, or trophoblasts). Other cells arecontemplated and consistent with the disclosure herein.

The biological sample can be extracted (e.g., biopsied) from anorganism, or obtained from a cell culture grown in liquid or in aculture dish. The biological sample comprises a sample that is fresh,frozen, fresh frozen, or archived (e.g., formalin-fixedparaffin-embedded; FFPE). The biological sample can be embedded in awax, resin, epoxy or agar. The biological sample can be fixed, forexample in any one or any combination of two or more of acetone,ethanol, methanol, formaldehyde, paraformaldehyde-Triton orglutaraldehyde. The biological sample can be sectioned or non-sectioned.The biological sample can be stained, de-stained or non-stained.

In some embodiments, the biological sample can be permeabilized afterbeing fixed to the surface described herein to permit the nucleic acidswithin the sample, including the target nucleic acid molecule, tomigrate from the cell(s) to the plurality of capture oligonucleotidesthat are immobilized to the surface. Permeabilization may allow an agent(such as a phospho-selective antibody, a nucleic acid conjugatedantibody, a nucleic acid probe, a primer, etc.) to enter into a cell andreach a concentration within the cell that is greater than that whichwould normally penetrate into the cell in the absence of suchpermeabilizing treatment. In some embodiments, cells may bepermeabilized in the presence of at least about 60%, 70%, 80%, 90% ormore methanol (or ethanol) and incubated on ice for a period of time.The period of time for incubation can be at least about 10, 15, 20, 25,30, 35, 40, 50, 60 or more minutes.

The biological sample can be permeabilized by contacting the biologicalsample with one or more permeabilizing agents, including organicsolvents, detergents, cross-linking agents and/or enzymes. In someembodiments, the organic solvents comprise acetone, ethanol, andmethanol. In some embodiments, the detergents comprise saponin, TritonX-100, Tween-20, or sodium dodecyl sulfate (SDS), or N-lauroylsarcosinesodium salt solution. In some embodiments, the cross-linking agentcomprises paraformaldehyde. In some embodiments, the enzyme comprisestrypsin, pepsin or protease (e.g. proteinase K). In some embodiments,the target nucleic acid molecule from the biological sample ishybridized (captured) on the capture oligonucleotides immobilized on thesupport in a manner that preserves spatial location information of thetarget nucleic acid molecule in the biological sample.

The biological sample can be utilized to generate a three-dimensionalpolymer matrix comprising the cellular and sub-cellular components(e.g., nucleic acid molecules) of the biological sample. Thethree-dimensional polymer matrix can be coupled to the surface describedherein, covalently or non-covalently. In some embodiments, thethree-dimensional polymer matrix is porous and comprises polymerized orcross-linked sub-cellular components, including the target nucleic acidmolecules. A polymer matrix may be formed within a biological sample(e.g., a cell or tissue) by flowing one or more polymer precursors(e.g., monomers, such as, for example, ethylene oxide for polyetheneglycol) into the biological sample and subjecting the one or morepolymer precursors to polymerization or cross-linking. Prior to, during,or subsequent to formation of the polymer matrix, positions of moieties(e.g., DNA, RNA, protein) within the biological sample may be fixed,using for example, a fixation agent (e.g., formaldehyde). A porousmatrix may be made according to various methods. For example, apolyacrylamide gel matrix can be polymerized with biotinylated DNAmolecules and acrydite-modified streptavidin monomers, using a suitableacrylamide:bis-acrylamide ratio to control the cross-linking density.Additional control over the molecular sieve size and density can beachieved by adding additional cross-linkers such as functionalizedpolyethylene glycols. Enablement for fixing biological sample to asurface, as well as generating a polymer matrix within a biologicalsample, is provided in PCT/US2019/055434, which is hereby incorporatedby reference in its entirety.

The biological sample comprises target nucleic acid molecule(s) that, insome cases, are analyzed using the systems, methods and compositionsdescribed herein. In some embodiments, the target nucleic acids comprisenaturally-occurring nucleic acids, recombinant nucleic acids and/orsynthesized nucleic acids. The target nucleic acid includes linearand/or circular forms. In some embodiments, the target nucleic acid maybe DNA. In some embodiments, the target nucleic acid may be genomic DNA.In some embodiments, the target nucleic acid may be viral DNA. In someembodiments, the target nucleic acid may be cell free DNA (cfDNA). Insome embodiments, the DNA is genomic DNA, methylated or un-methylatedDNA, and/or organellar DNA. The DNA can be fragmented and/orunfragmented. In some embodiments, the target nucleic acid molecule(s)comprise RNA, including poly-A RNA and/or non-poly-a RNA. The RNAcomprises coding and/or non-coding RNA. The RNA comprises tRNA, rRNA,small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA(miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA),antisense RNA, non-coding RNA and/or protein-encoding RNA.

The target nucleic acids of the instant disclosure have a fixedthree-dimensional relationship with the biological sample after thebiological sample is coupled to the surface. This fixedthree-dimensional relationship, at least partially, enables theidentification of spatial and cellular origin within the biologicalsample following nucleic acid identification using the systems andmethods described herein.

Target Nucleic Acid Capture and Preparation. Provided herein are methodsof hybridizing the target nucleic acid to the capture oligonucleotidescoupled to the surface (e.g., low non-specific binding surface) in thepresence of the biological sample. In some cases, hybridization bufferformulations described which, in combination with the disclosedlow-binding supports, provide for improved hybridization rates,hybridization specificity (or stringency), and hybridization efficiency(or yield). As used herein, hybridization specificity is a measure ofthe ability of tethered adapter sequences, primer sequences, oroligonucleotide sequences in general to correctly hybridize only tocompletely complementary sequences, while hybridization efficiency is ameasure of the percentage of total available tethered adapter sequences,primer sequences, or oligonucleotide sequences in general that arehybridized to complementary sequences.

Improved hybridization specificity and/or efficiency may be achievedthrough optimization of the hybridization buffer formulation used withthe disclosed low-binding surfaces, and will be discussed in more detailin the examples below. Examples of hybridization buffer components thatmay be adjusted to achieve improved performance include, but are notlimited to, buffer type, organic solvent mixtures, buffer pH, bufferviscosity, detergents and zwitterionic components, ionic strength(including adjustment of both monovalent and divalent ionconcentrations), antioxidants and reducing agents, carbohydrates, BSA,polyethylene glycol, dextran sulfate, betaine, other additives, and thelike.

By way of non-limiting example, suitable buffers for use in formulatinga hybridization buffer may include, but are not limited to, phosphatebuffered saline (PBS), succinate, citrate, histidine, acetate, Tris,TAPS, MOPS, PIPES, HEPES, IVIES, and the like. The choice of appropriatebuffer will generally be dependent on the target pH of the hybridizationbuffer solution. In general, the desired pH of the buffer solution willrange from about pH 4 to about pH 8.4. In some embodiments, the bufferpH may be at least 4.0, at least 4.5, at least 5.0, at least 5.5, atleast 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, atleast 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, atleast 8.0, at least 8.2, or at least 8.4. In some embodiments, thebuffer pH may be at most 8.4, at most 8.2, at most 8.0, at most 7.8, atmost 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at most6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at most 5.0, atmost 4.5, or at most 4.0. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances, the desired pH mayrange from about 6.4 to about 7.2. Those of skill in the art willrecognize that the buffer pH may have any value within this range, forexample, about 7.25.

Suitable detergents for use in hybridization buffer formulation include,but are not limited to, zitterionic detergents (e.g.,1-Dodecanoyl-sn-glycero-3-phosphocholine,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,NDimethylmyristylammonio) propanesulfonate, ASB-C80, C7BzO, CHAPS,CHAPS hydrate, CHAPSO, DDMAB, Dimethylethylammoniumpropane sulfonate,N,N-Dimethyldodecylamine Noxide,N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, orN-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and anionic,cationic, and non-ionic detergents. Examples of nonionic detergentsinclude poly(oxyethylene) ethers and related polymers (e.g. Brij®,TWEEN®, TRITON®, TRITON X-100 and IGEPAL® CA-630), bile salts, andglycosidic detergents.

The use of the disclosed low non-specific binding supports either aloneor in combination with optimized buffer formulations may yield relativehybridization rates that range from about 2× to about 20× faster thanthat for a conventional hybridization protocol. In some instances, therelative hybridization rate may be at least 2×, at least 3×, at least4×, at least 5×, at least 6×, at least 7×, at least 8×, at least 9×, atleast 10×, at least 12×, at least 14×, at least 16×, at least 18×, atleast 20×, at least 25×, at least 30×, or at least 40× that for aconventional hybridization protocol.

The use of the disclosed low non-specific binding supports alone or incombination with optimized buffer formulations may yield totalhybridization reaction times (i.e., the time required to reach 90%, 95%,98%, or 99% completion of the hybridization reaction) of less than 60minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10minutes, or 5 minutes for any of these completion metrics.

The use of the disclosed low non-specific binding supports alone or incombination with optimized buffer formulations may yield improvedhybridization specificity compared to that for a conventionalhybridization protocol. In some embodiments, the hybridizationspecificity that may be achieved is better than 1 base mismatch in 10hybridization events, 1 base mismatch in 20 hybridization events, 1 basemismatch in 30 hybridization events, 1 base mismatch in 40 hybridizationevents, 1 base mismatch in 50 hybridization events, 1 base mismatch in75 hybridization events, 1 base mismatch in 100 hybridization events, 1base mismatch in 200 hybridization events, 1 base mismatch in 300hybridization events, 1 base mismatch in 400 hybridization events, 1base mismatch in 500 hybridization events, 1 base mismatch in 600hybridization events, 1 base mismatch in 700 hybridization events, 1base mismatch in 800 hybridization events, 1 base mismatch in 900hybridization events, 1 base mismatch in 1,000 hybridization events, 1base mismatch in 2,000 hybridization events, 1 base mismatch in 3,000hybridization events, 1 base mismatch in 4,000 hybridization events, 1base mismatch in 5,000 hybridization events, 1 base mismatch in 6,000hybridization events, 1 base mismatch in 7,000 hybridization events, 1base mismatch in 8,000 hybridization events, 1 base mismatch in 9,000hybridization events, or 1 base mismatch in 10,000 hybridization events.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized buffer formulations mayyield improved hybridization efficiency (e.g., the fraction of availableoligonucleotide primers on the support surface that are successfullyhybridized with target oligonucleotide sequences) compared to that for aconventional hybridization protocol. In some instances, thehybridization efficiency that may be achieved is better than 50%, 60%,70%, 80%, 85%, 90%, 95%, 98%, or 99% for any of the input targetoligonucleotide concentrations specified below and in any of thehybridization reaction times specified above. In some instances, e.g.,wherein the hybridization efficiency is less than 100%, the resultingsurface density of target nucleic acid sequences hybridized to thesupport surface may be less than the surface density of oligonucleotideadapter or primer sequences on the surface.

In some instances, use of the disclosed low non-specific bindingsupports for nucleic acid hybridization (or amplification) applicationsusing conventional hybridization (or amplification) protocols, oroptimized hybridization (or amplification) protocols may lead to areduced requirement for the input concentration of target (or sample)nucleic acid molecules contacted with the support surface. For example,in some instances, the target (or sample) nucleic acid molecules may becontacted with the support surface at a concentration ranging from about10 pM to about 1 μM (i.e., prior to annealing or amplification). In someinstances, the target (or sample) nucleic acid molecules may beadministered at a concentration of at least 10 pM, at least 20 pM, atleast 30 pM, at least 40 pM, at least 50 pM, at least 100 pM, at least200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, atleast 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, atleast 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, atleast 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, atleast 900 nM, or at least 1 μM. In some instances, the target (orsample) nucleic acid molecules may be administered at a concentration ofat most 1 μM, at most 900 nM, at most 800 nm, at most 700 nM, at most600 nM, at most 500 nM, at most 400 nM, at most 300 nM, at most 200 nM,at most 100 nM, at most 90 nM, at most 80 nM, at most 70 nM, at most 60nM, at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, at most10 nM, at most 1 nM, at most 900 pM, at most 800 pM, at most 700 pM, atmost 600 pM, at most 500 pM, at most 400 pM, at most 300 pM, at most 200pM, at most 100 pM, at most 90 pM, at most 80 pM, at most 70 pM, at most60 pM, at most 50 pM, at most 40 pM, at most 30 pM, at most 20 pM, or atmost 10 pM. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the target (or sample)nucleic acid molecules may be administered at a concentration rangingfrom about 90 pM to about 200 nM. Those of skill in the art willrecognize that the target (or sample) nucleic acid molecules may beadministered at a concentration having any value within this range,e.g., about 855 nM.

In another example, a volume of the biological sample that may becontacted with the surface may be reduced relative to a comparablebiological sample analyzed using a comparable surface using standardhybridization reagents. In some embodiments, a fluid sample comprisingthe target (or sample) nucleic acid molecules may be in a range ofsample volumes that is about 5 μl to about 900 μl. In some instances,the range of sample volumes is about 5 μl to about 800 μl. In someinstances, the range of sample volumes is about 5 μl to about 700 μl. Insome instances, the range of sample volumes is about 5 μl to about 600μl. In some instances, the range of sample volumes is about 5 μl toabout 500 μl. In some instances, the range of sample volumes is about 5μl to about 400 μl. In some instances, the range of sample volumes isabout 5 μl to about 300 μl. In some instances, the range of samplevolumes is about 5 μl to about 200 In some instances, the range ofsample volumes is about 5 μl to about 150 μl. In some instances, therange of sample volumes is 5 μl to about 100 μl. In some instances, therange of sample volumes is about 5 μl to about 90 μl. In some instances,the range of sample volumes is about 5 μl to about 85 μl. In someinstances, the range of sample volumes is about 5 μl to about 80 μl. Insome instances, the range of sample volumes is about 5 μl to about 75μl. In some instances, the range of sample volumes is about 5 μl toabout 70 μl. In some instances, the range of sample volumes is about 5μl to about 65 μl. In some instances, the range of sample volumes isabout 5 μl to about 60 μl. In some instances, the range of samplevolumes is about 5 μl to about 55 μl. In some instances, the range ofsample volumes is about 5 μl to about 50 μl. In some instances, therange of sample volumes is about 15 μl to about 150 μl. In someinstances, the range of sample volumes is about 15 μl to about 120 μl.In some instances, the range of sample volumes is 15 μl to about 100 μl.In some instances, the range of sample volumes is about 15 μl to about90 In some instances, the range of sample volumes is about 15 μl toabout 85 μl. In some instances, the range of sample volumes is about 15μl to about 80 μl. In some instances, the range of sample volumes isabout 15 μl to about 75 μl. In some instances, the range of samplevolumes is about 15 μl to about 70 μl. In some instances, the range ofsample volumes is about 15 μl to about 65 μl. In some instances, therange of sample volumes is about 15 μl to about 60 μl. In someinstances, the range of sample volumes is about 15 μl to about 55 μl. Insome instances, the range of sample volumes is about 15 μl to about 50μl.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized hybridization bufferformulations may result in a surface density of hybridized target (orsample) oligonucleotide molecules (i.e., prior to performing anysubsequent solid-phase or clonal amplification reaction) ranging fromabout from about 0.0001 target oligonucleotide molecules per μm2 toabout 1,000,000 target oligonucleotide molecules per μm2. In someinstances, the surface density of hybridized target oligonucleotidemolecules may be at least 0.0001, at least 0.0005, at least 0.001, atleast 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5,at least 1, at least 5, at least 10, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 600, at least 700, at least 800, at least 900, at least 1,000, atleast 1,500, at least 2,000, at least 2,500, at least 3,000, at least3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500,at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000,at least 15,000, at least 20,000, at least 25,000, at least 30,000, atleast 35,000, at least 40,000, at least 45,000, at least 50,000, atleast 55,000, at least 60,000, at least 65,000, at least 70,000, atleast 75,000, at least 80,000, at least 85,000, at least 90,000, atleast 95,000, at least 100,000, at least 150,000, at least 200,000, atleast 250,000, at least 300,000, at least 350,000, at least 400,000, atleast 450,000, at least 500,000, at least 550,000, at least 600,000, atleast 650,000, at least 700,000, at least 750,000, at least 800,000, atleast 850,000, at least 900,000, at least 950,000, or at least 1,000,000molecules per μm2. In some instances, the surface density of hybridizedtarget oligonucleotide molecules may be at most 1,000,000, at most950,000, at most 900,000, at most 850,000, at most 800,000, at most750,000, at most 700,000, at most 650,000, at most 600,000, at most550,000, at most 500,000, at most 450,000, at most 400,000, at most350,000, at most 300,000, at most 250,000, at most 200,000, at most150,000, at most 100,000, at most 95,000, at most 90,000, at most85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000,at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000,at most 15,000, at most 10,000, at most 9,500, at most 9,000, at most8,500, at most 8,000, at most 7,500, at most 7,000, at most 6,500, atmost 6,000, at most 5,500, at most 5,000, at most 4,500, at most 4,000,at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most1,500, at most 1,000, at most 900, at most 800, at most 700, at most600, at most 500, at most 400, at most 300, at most 200, at most 100, atmost 90, at most 80, at most 70, at most 60, at most 50, at most 40, atmost 30, at most 20, at most 10, at most 5, at most 1, at most 0.5, atmost 0.1, at most 0.05, at most 0.01, at most 0.005, at most 0.001, atmost 0.0005, or at most 0.0001 molecules per μm2. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe surface density of hybridized target oligonucleotide molecules mayrange from about 3,000 molecules per μm2 to about 20,000 molecules perμm2. Those of skill in the art will recognize that the surface densityof hybridized target oligonucleotide molecules may have any value withinthis range, e.g., about 2,700 molecules per μm2.

Stated differently, in some instances the use of the disclosed lownon-specific binding supports alone or in combination with optimizedhybridization buffer formulations may result in a surface density ofhybridized target (or sample) oligonucleotide molecules (i.e., prior toperforming any subsequent solid-phase or clonal amplification reaction)ranging from about 100 hybridized target oligonucleotide molecules permm2 to about 1×107 oligonucleotide molecules per mm2 or from about 100hybridized target oligonucleotide molecules per mm2 to about 1×1012hybridized target oligonucleotide molecules per mm2. In some instances,the surface density of hybridized target oligonucleotide molecules maybe at least 100, at least 500, at least 1,000, at least 4,000, at least5,000, at least 6,000, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, at least 1,000,000, at least 5,000,000, atleast 1×107, at least 5×107, at least 1×108, at least 5×108, at least1×109, at least 5×109, at least 1×1010, at least 5×1010, at least1×1011, at least 5×1011, or at least 1×1012 molecules per mm2. In someinstances, the surface density of hybridized target oligonucleotidemolecules may be at most 1×1012, at most 5×1011, at most 1×1011, at most5×1010, at most 1×1010, at most 5×109, at most 1×109, at most 5×108, atmost 1×108, at most 5×107, at most 1×107, at most 5,000,000, at most1,000,000, at most 950,000, at most 900,000, at most 850,000, at most800,000, at most 750,000, at most 700,000, at most 650,000, at most600,000, at most 550,000, at most 500,000, at most 450,000, at most400,000, at most 350,000, at most 300,000, at most 250,000, at most200,000, at most 150,000, at most 100,000, at most 95,000, at most90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000,at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000,at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most1,000, at most 500, or at most 100 molecules per mm2. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the surface density of hybridized target oligonucleotidemolecules may range from about 5,000 molecules per mm2 to about 50,000molecules per mm2. Those of skill in the art will recognize that thesurface density of hybridized target oligonucleotide molecules may haveany value within this range, e.g., about 50,700 molecules per mm2.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) hybridized to the oligonucleotide adapter orprimer molecules attached to the low-binding support surface may rangein length from about 0.02 kilobases (kb) to about 20 kb or from about0.1 kilobases (kb) to about 20 kb. In some instances, the targetoligonucleotide molecules may be at least 0.001 kb, at least 0.005 kb,at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb inlength, at least 0.2 kb in length, at least 0.3 kb in length, at least0.4 kb in length, at least 0.5 kb in length, at least 0.6 kb in length,at least 0.7 kb in length, at least 0.8 kb in length, at least 0.9 kb inlength, at least 1 kb in length, at least 2 kb in length, at least 3 kbin length, at least 4 kb in length, at least 5 kb in length, at least 6kb in length, at least 7 kb in length, at least 8 kb in length, at least9 kb in length, at least 10 kb in length, at least 15 kb in length, atleast 20 kb in length, at least 30 kb in length, or at least 40 kb inlength, or any intermediate value spanned by the range described herein,e.g., at least 0.85 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-stranded,multimeric nucleic acid molecules further comprising repeats of aregularly occurring monomer unit. In some instances, the single-strandedor double-stranded, multimeric nucleic acid molecules may be at least0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, atleast 0.05 kb, at least 0.1 kb in length, at least 0.2 kb in length, atleast 0.3 kb in length, at least 0.4 kb in length, at least 0.5 kb inlength, at least 1 kb in length, at least 2 kb in length, at least 3 kbin length, at least 4 kb in length, at least 5 kb in length, at least 6kb in length, at least 7 kb in length, at least 8 kb in length, at least9 kb in length, at least 10 kb in length, at least 15 kb in length, orat least 20 kb in length, at least 30 kb in length, or at least 40 kb inlength, or any intermediate value spanned by the range described herein,e.g., about 2.45 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-strandedmultimeric nucleic acid molecules comprising from about 2 to about 100copies of a regularly repeating monomer unit. In some instances, thenumber of copies of the regularly repeating monomer unit may be at least2, at least 3, at least 4, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 55, at least 60, at least 65, at least 70, atleast 75, at least 80, at least 85, at least 90, at least 95, and atleast 100. In some instances, the number of copies of the regularlyrepeating monomer unit may be at most 100, at most 95, at most 90, atmost 85, at most 80, at most 75, at most 70, at most 65, at most 60, atmost 55, at most 50, at most 45, at most 40, at most 35, at most 30, atmost 25, at most 20, at most 15, at most 10, at most 5, at most 4, atmost 3, or at most 2. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the number of copiesof the regularly repeating monomer unit may range from about 4 to about60. Those of skill in the art will recognize that the number of copiesof the regularly repeating monomer unit may have any value within thisrange, e.g., about 17. Thus, in some instances, the surface density ofhybridized target sequences in terms of the number of copies of a targetsequence per unit area of the support surface may exceed the surfacedensity of oligonucleotide primers even if the hybridization efficiencyis less than 100%.

As used herein, the phrase “nucleic acid surface amplification” (NASA)is used interchangeably with the phrase “solid-phase nucleic acidamplification” (or simply “solid-phase amplification”). In some aspectsof the present disclosure, nucleic acid amplification formulations aredescribed which, in combination with the disclosed low-binding supports,provide for improved amplification rates, amplification specificity, andamplification efficiency. As used herein, specific amplification refersto amplification of template library oligonucleotide strands that havebeen tethered to the solid support either covalently or non-covalently.As used herein, non-specific amplification refers to amplification ofprimer-dimers or other non-template nucleic acids. As used herein,amplification efficiency is a measure of the percentage of tetheredoligonucleotides on the support surface that are successfully amplifiedduring a given amplification cycle or amplification reaction. Nucleicacid amplification performed on surfaces disclosed herein may obtainamplification efficiencies of at least 50%, 60%, 70%, 80%, 90%, 95%, orgreater than 95%, such as 98% or 99%.

Any of a variety of thermal cycling or isothermal nucleic acidamplification schemes may be used with the disclosed low-bindingsupports. Examples of nucleic acid amplification methods that may beutilized with the disclosed low non-specific binding supports include,but are not limited to, polymerase chain reaction (PCR), multipledisplacement amplification (MDA), transcription-mediated amplification(TMA), nucleic acid sequence-based amplification (NASBA), stranddisplacement amplification (SDA), real-time SDA, bridge amplification,isothermal bridge amplification, rolling circle amplification,circle-to-circle amplification, helicase-dependent amplification,recombinase-dependent amplification, or single-stranded binding (SSB)protein-dependent amplification.

In some embodiments, a rolling circle amplification reaction comprises:(1) forming a trapped nucleotide-polymerase complexes by contacting aplurality of immobilized covalently closed circular nucleic acidmolecules with (i) a first plurality of polymerases having stranddisplacement activity; (ii) a plurality of nucleotides (e.g., one typeof nucleotide or, a mixture of dATP, dGTP, dCTP and dTTP); (iii) anon-catalytic divalent cation that mediates nucleotide binding but notnucleotide incorporation (e.g., strontium or barium), and optionally(iv) a plurality of amplification primers if the covalently closedcircular molecules lack a primer. The rolling circle amplificationreaction further comprises: (4) conducting a nucleotide polymerizationreaction by contacting the trapped nucleotide-polymerase complex with(i) at least one divalent cation that mediates nucleotide binding andmediates nucleotide incorporation (e.g., magnesium and/or manganese),and (ii) a second plurality of nucleotides (e.g., a mixture of dATP,dGTP, dCTP and dTTP), under a condition suitable for conducting anisothermal rolling circle amplification reaction to generate a pluralityof immobilized concatemers.

In some embodiments, the rolling circle amplification reaction furthercomprises a plurality of compaction oligonucleotides that can hybridizeto portions of the concatemer to collapse the concatemer into a morecompact shape and size. the compaction oligonucleotide is asingle-stranded nucleic acid molecule having two identical sequencesseparated by a short linker sequence, where the two identical sequencesare reverse-complementary to a portion of the concatemer. The compactionoligonucleotide can be any length, for example 20-100 nucleotides. Thetwo identical sequence regions hybridize to the concatemer to pulltogether distal portions of the concatemer causing compaction of theconcatemer. In some embodiments, the compaction oligonucleotide isresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the compactionoligonucleotide comprises any one or any combination of two or more of:3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base.

In some embodiments, in the trapped nucleotide-polymerase mixture ofstep (c), the first plurality of polymerases having strand displacementactivity comprise phi29 DNA polymerase, large fragment of Bst DNApolymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNApolymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase,M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or DeepVent DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNApolymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, in the amplification primers comprisesingle-stranded nucleic acid primers having a length of about 5-25nucleotides. In some embodiments, the amplification primers areresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the amplification primerscomprise any one or any combination of two or more of: 3′ terminal endphosphorylation; at least two 3′ terminal end nucleotides having aphosphorothioate bond therebetween; at least one 3′ terminal endnucleotide having a 2′-O-methyl moiety; and/or at least one 3′ terminalnucleotide having a 2′ fluoro base.

In some embodiments, the rolling circle amplification reaction furthercomprises at least one accessory protein or enzyme, including helicase,single-stranded binding (SSB) protein, or recombinase (e.g., T4 uvsX)and/or recombinase accessory factor (e.g., T4 uvsY or T4 gp32).

In some embodiments, the isothermal rolling circle amplificationreaction can be conducted at a temperature of about 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40° C.

In some embodiments, the concatemer can contain at least 2, 10, 100,200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,or more copies of the repeat units.

The rolling circle amplification method can be followed by a multipledisplacement amplification reaction which employs random-sequenceprimers. The multiple displacement amplification reaction comprises: (1)forming a multiple displacement amplification (MDA) reaction mixture bycontacting the plurality of immobilized concatemers with (i) a secondplurality of polymerases having strand displacement activity, and (ii) aplurality of soluble amplification primers wherein individualamplification primers in the plurality are exonuclease-resistant andhave a 3′ extendible end and comprise a random sequence that canhybridize to a portion of the single-stranded circular nucleic acidtemplates, (iii) a second plurality of nucleotides (e.g., a mixture ofdATP, dGTP, dCTP and dTTP), and (iv) at least one divalent cation thatmediates nucleotide binding and mediates nucleotide incorporation (e.g.,magnesium and/or manganese); and (2) conducting an isothermal multipledisplacement amplification (MDA) reaction to generate a plurality ofimmobilized branched concatemers.

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the second plurality of polymerases having stranddisplacement activity comprises phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-)DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), orvariant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific),or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the plurality of amplification primers comprisesingle-stranded nucleic acid primers having a length of about 5-25nucleotides. In some embodiments, the plurality of soluble amplificationprimers comprise non-protected single-stranded nucleic acid primers. Insome embodiments, the plurality of soluble amplification primerscomprise protected single-stranded nucleic acid primers that areresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the plurality of solubleamplification primers comprise any one or any combination of two or moreof: 3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base. In some embodiments, theplurality of soluble amplification primers comprise a population ofprimers having the same length, for example a length of 6 or 9nucleotides. In some embodiments, the plurality of soluble amplificationprimers comprise a population of primers having a mixture of differentlengths, for example a mixture comprising 6-mer and 9-mer primers. Insome embodiments, the plurality of soluble amplification primerscomprise a mixture of primers having random sequences including up to 4⁶different sequences (e.g., for the 6-mers) or 4⁹ different sequences(e.g., for the 9-mers).

In some embodiments, the multiple displacement amplification (MDA)reaction mixture can further comprise at least one accessory protein orenzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the isothermal multiple displacement amplification(MDA) reaction can be conducted at a temperature of about 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45° C.

The rolling circle amplification method can be followed by a multipledisplacement amplification reaction which employs a primase-polymeraseenzyme. The multiple displacement amplification reaction comprises: (1)forming a multiple displacement amplification (MDA) reaction mixture bycontacting the plurality of immobilized concatemers with (1) a secondplurality of polymerases having strand displacement activity, (ii) aplurality of DNA primase-polymerase enzymes, (iii) a second plurality ofnucleotides (e.g., a mixture of dATP, dGTP, dCTP and dTTP), and (iv) atleast one divalent cation that mediates nucleotide binding and mediatesnucleotide incorporation (e.g., magnesium and/or manganese), and (2)conducting an isothermal multiple displacement amplification (MDA)reaction to generate a plurality of immobilized branched concatemers. Insome embodiments, the multiple displacement amplification reaction isconducted without added amplification primers (e.g., a primerlessreaction).

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the second plurality of polymerases having stranddisplacement activity comprises phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-)DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), orvariant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific),or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, the plurality of DNA primase-polymerase enzymescomprise an enzyme from Thermus thermophilus HB27 (e.g., Tth PrimPolenzyme).

In some embodiments, the multiple displacement amplification (MDA)reaction mixture further comprises at least one accessory protein orenzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the isothermal multiple displacement amplification(MDA) reaction can be conducted at a temperature of about 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45° C.

Another embodiment of the two stage amplification methods includesexposing the concatemer to nucleic acid relaxing agents (first stage)and then conducting a flexing amplification reaction during the secondstage. Without wishing to be bound by theory, it is postulated that thenucleic acid relaxing agent(s) can disrupt hydrogen bonding (e.g.,denaturation) in the plurality of immobilized nucleic acid concatemerswhich causes the structure of the nucleic acid concatemers to relax andincreases the number of new duplex formations between the immobilizedsurface capture primers and portions of the nucleic acid concatemers,thereby increasing the opportunity to generate new concatemers from theduplexed immobilized surface capture primers. The new concatemers can begenerated during the flexing amplification reaction. The inclusion ofthe relaxing agents can cause nucleic acid denaturation without use ofdenaturation temperatures or denaturation chemicals.

In some embodiments, the amplification method comprises: (1) conductingan on-support rolling circle amplification to generate a plurality ofsingle-stranded concatemers, (2) forming a relaxant reaction mixture,(3) forming a flexing amplification reaction mixture, (4) conducting aflexing amplification reaction on the support (e.g., with no addedsoluble primers) to generate a plurality of double-stranded concatemers,(5) washing, and (6) repeating steps (2)-(5) at least once.

In some embodiments, the relaxant reaction mixture of step (2) can beformed with at least one nucleic acid relaxing agent that can disrupthydrogen bonding in the immobilized nucleic acid concatemers. Exemplaryrelaxing agents include nucleic acid denaturants, chaotropic compounds,amide compounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents includesodium iodide, potassium iodide and polyamines

In some embodiments, the relaxant reaction mixture comprises any one ora combination of two or more of a group selected from urea, guanidinehydrochloride, guanidine thiocyanate, formamide, acetamide,NN-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide),1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol,1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethyoxyethane,2-methoxyethanol, sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxant reaction mixture comprises formamideand SSC. In some embodiments, the relaxant reaction mixture comprisesacetonitrile, formamide and SSC. In some embodiments, the relaxantreaction mixture comprises acetonitrile, formamide and IVIES(2-(4-morpholino)-ethane sulfonic acid). In some embodiments, therelaxant reaction mixture comprises acetonitrile, formamide, guanidiumhydrochloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid). In some embodiments, the relaxant reaction mixture comprisesacetonitrile, formamide, urea and HEPES. In some embodiments, the SSC inthe relaxant reaction mixture can be 1×, 2×, 3× or 4×.

In some embodiments, in the forming the relaxant reaction mixture ofstep (2), the temperature ramp-up condition can be conducted from about20° C. to about 70° C., the relaxant incubation condition can beconducted at a temperature of about 40-70° C., and the temperatureramp-down condition can be conducted from about 70° C. to about 20° C. Askilled artisan will recognize that the temperature ramp-up, relaxantincubation temperature, and temperature ramp-down conditions can bemodified.

In some embodiments, in the flexing amplification reaction mixture ofstep (3), the second plurality of polymerases having strand displacementactivity comprises large fragment of Bst DNA polymerase (e.g.,exonuclease minus), phi29 DNA polymerase, large fragment of Bsu DNApolymerase, and Bca (exo-) DNA polymerase, Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNApolymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi fromExpedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo FisherScientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, in the flexing amplification reaction mixture ofstep (2), the concentration (e.g., total concentration) of the thirdplurality of nucleotides can promote a nucleotide polymerizationreaction. For example, the concentration (e.g., total concentration) ofthe third plurality of nucleotides is about 0.1-10 mM.

In some embodiments, the third plurality of nucleotides in the flexingamplification reaction mixture of step (2) comprise a mixture of two ormore nucleotides selected from a group consisting of dATP, dGTP, dCTPand dTTP.

In some embodiments, in the flexing amplification reaction mixture ofstep (2), the at least one divalent cation that mediates nucleotidebinding and mediates nucleotide polymerization comprises a catalyticdivalent cation. In some embodiments, the catalytic divalent cationcomprises magnesium and/or manganese. The concentration of the catalyticdivalent cation in the amplification reaction mixture can be about 1-20mM.

In some embodiments, the flexing amplification reaction mixture of step(2) can include at least one accessory protein or enzyme, includinghelicase, single-stranded binding (SSB) protein, or recombinase (e.g.,T4 uvsX) and/or recombinase accessory factor (e.g., T4 uvsY or T4 gp32).In some embodiments, these accessory proteins can be omitted.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-up condition can be conducted from about 20° C. toabout 90° C. In some embodiments, in the flexing amplification reactionof step (4), the temperature ramp-up condition can be conducted forabout 5-15 seconds, or about 15-30 seconds, or about 30-45 seconds, orabout 45-60 seconds, or longer. In some embodiments, in the flexingamplification reaction of step (4), the amplification incubationcondition can be about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69 or 70° C., or at a higher temperature. Insome embodiments, in the flexing amplification reaction of step (4), theamplification incubation condition can be conducted for about 30-45seconds, or about 45-60 seconds, or about 60-75 seconds, or about 75-90seconds, or longer. In some embodiments, in the flexing amplificationreaction of step (4), the temperature ramp-down condition can beconducted from about 90° C. to about 20° C.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-down condition can be conducted for about 5-15seconds, or about 15-30 seconds, or about 30-45 seconds, or about 45-60seconds, or longer. In some embodiments, in the washing of step (5), thewash buffer comprises 1×SSC, or 1×SSC with cobalt hexamine. In someembodiments, steps (2)-(5) can be repeated at least once, or repeated upto 10 times, or repeated up to 15 times, or repeated up to 20 times, orrepeated up to 30 times or more.

Often, improvements in amplification rate, amplification specificity,and amplification efficiency may be achieved using the disclosed lownon-specific binding supports alone or in combination with formulationsof the amplification reaction components. In addition to inclusion ofnucleotides, one or more polymerases, helicases, single-stranded bindingproteins, etc. (or any combination thereof), the amplification reactionmixture may be adjusted in a variety of ways to achieve improvedperformance including, but are not limited to, choice of buffer type,buffer pH, organic solvent mixtures, buffer viscosity, detergents andzwitterionic components, ionic strength (including adjustment of bothmonovalent and divalent ion concentrations), antioxidants and reducingagents, carbohydrates, BSA, polyethylene glycol, dextran sulfate,betaine, other additives, and the like.

The use of the disclosed low non-specific binding supports alone or incombination with optimized amplification reaction formulations may yieldincreased amplification rates compared to those obtained usingconventional supports and amplification protocols. In some instances,the relative amplification rates that may be achieved may be at least2×, at least 3×, at least 4×, at least 5×, at least 6×, at least 7×, atleast 8×, at least 9×, at least 10×, at least 12×, at least 14×, atleast 16×, at least 18×, or at least 20× that for use of conventionalsupports and amplification protocols for any of the amplificationmethods described above.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized buffer formulations mayyield total amplification reaction times (i.e., the time required toreach 90%, 95%, 98%, or 99% completion of the amplification reaction) ofless than 180 mins, 120 mins, 90 min, 60 minutes, 50 minutes, 40minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3minutes, 1 minute, 50 s, 40 s, 30 s, 20 s, or 10 s for any of thesecompletion metrics.

Some low-binding support surfaces disclosed herein exhibit a ratio ofspecific binding to nonspecific binding of a fluorophore such as Cy3 ofat least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1,75:1, 100:1, or greater than 100:1, or any intermediate value spanned bythe range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to nonspecific fluorescence signal for a fluorophore such asCy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediatevalue spanned by the range herein.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification bufferformulations may enable faster amplification reaction times (i.e., thetimes required to reach 90%, 95%, 98%, or 99% completion of theamplification reaction) of no more than 60 minutes, 50 minutes, 40minutes, 30 minutes, 20 minutes, or 10 minutes. Similarly, use of thedisclosed low non-specific binding supports alone or in combination withoptimized buffer formulations may enable amplification reactions to becompleted in some cases in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or no more than 30 cycles.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification reactionformulations may yield increased specific amplification and/or decreasednon-specific amplification compared to that obtained using conventionalsupports and amplification protocols. In some instances, the resultingratio of specific amplification-to-non-specific amplification that maybe achieved is at least 4:1 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1,40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1,600:1, 700:1, 800:1, 900:1, or 1,000:1.

In some instances, the use of the low non-specific binding supportsalone or in combination with optimized amplification reactionformulations may yield increased amplification efficiency compared tothat obtained using conventional supports and amplification protocols.In some instances, the amplification efficiency that may be achieved isbetter than 50%, 60%, 70% 80%, 85%, 90%, 95%, 98%, or 99% in any of theamplification reaction times specified above.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) hybridized to theoligonucleotide adapter or primer molecules attached to the low-bindingsupport surface may range in length from about 0.02 kilobases (kb) toabout 20 kb or from about 0.1 kilobases (kb) to about 20 kb. In someinstances, the clonally-amplified target oligonucleotide molecules maybe at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb inlength, at least 0.3 kb in length, at least 0.4 kb in length, at least0.5 kb in length, at least 1 kb in length, at least 2 kb in length, atleast 3 kb in length, at least 4 kb in length, at least 5 kb in length,at least 6 kb in length, at least 7 kb in length, at least 8 kb inlength, at least 9 kb in length, at least 10 kb in length, at least 15kb in length, or at least 20 kb in length, or any intermediate valuespanned by the range described herein, e.g., at least 0.85 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded, multimeric nucleic acid moleculesfurther comprising repeats of a regularly occurring monomer unit. Insome instances, the clonally-amplified single-stranded ordouble-stranded, multimeric nucleic acid molecules may be at least 0.1kb in length, at least 0.2 kb in length, at least 0.3 kb in length, atleast 0.4 kb in length, at least 0.5 kb in length, at least 1 kb inlength, at least 2 kb in length, at least 3 kb in length, at least 4 kbin length, at least 5 kb in length, at least 6 kb in length, at least 7kb in length, at least 8 kb in length, at least 9 kb in length, at least10 kb in length, at least 15 kb in length, or at least 20 kb in length,or any intermediate value spanned by the range described herein, e.g.,about 2.45 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded multimeric nucleic acid moleculescomprising from about 2 to about 100 copies of a regularly repeatingmonomer unit. In some instances, the number of copies of the regularlyrepeating monomer unit may be at least 2, at least 3, at least 4, atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, and at least 100. In some instances, thenumber of copies of the regularly repeating monomer unit may be at most100, at most 95, at most 90, at most 85, at most 80, at most 75, at most70, at most 65, at most 60, at most 55, at most 50, at most 45, at most40, at most 35, at most 30, at most 25, at most 20, at most 15, at most10, at most 5, at most 4, at most 3, or at most 2. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe number of copies of the regularly repeating monomer unit may rangefrom about 4 to about 60. Those of skill in the art will recognize thatthe number of copies of the regularly repeating monomer unit may haveany value within this range, e.g., about 12. Thus, in some instances,the surface density of clonally-amplified target sequences in terms ofthe number of copies of a target sequence per unit area of the supportsurface may exceed the surface density of oligonucleotide primers evenif the hybridization and/or amplification efficiencies are less than100%.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification reactionformulations may yield increased clonal copy number compared to thatobtained using conventional supports and amplification protocols. Insome instances, e.g., wherein the clonally-amplified target (or sample)oligonucleotide molecules comprise concatenated, multimeric repeats of amonomeric target sequence, the clonal copy number may be substantiallysmaller than compared to that obtained using conventional supports andamplification protocols. Thus, in some instances, the clonal copy numbermay range from about 1 molecule to about 100,000 molecules (e.g., targetsequence molecules) per amplified colony. In some instances, the clonalcopy number may be at least 1, at least 5, at least 10, at least 50, atleast 100, at least 500, at least 1,000, at least 2,000, at least 3,000,at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least8,000, at least 9,000, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, or at least100,000 molecules per amplified colony. In some instances, the clonalcopy number may be at most 100,000, at most 95,000, at most 90,000, atmost 85,000, at most 80,000, at most 75,000, at most 70,000, at most65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000,at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most20,000, at most 15,000, at most 10,000, at most 9,000, at most 8,000, atmost 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000,at most 2,000, at most 1,000, at most 500, at most 100, at most 50, atmost 10, at most 5, or at most 1 molecule per amplified colony. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the clonal copy number may range from about 2,000molecules to about 9,000 molecules. Those of skill in the art willrecognize that the clonal copy number may have any value within thisrange, e.g., about 2,220 molecules in some instances, or about 2molecules in others.

As noted above, in some instances the amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may compriseconcatenated, multimeric repeats of a monomeric target sequence. In someinstances, the amplified target (or sample) oligonucleotide molecules(or nucleic acid molecules) may comprise a plurality of molecules eachof which comprises a single monomeric target sequence. Thus, the use ofthe disclosed low non-specific binding supports alone or in combinationwith optimized amplification reaction formulations may result in asurface density of target sequence copies that ranges from about 100target sequence copies per mm2 to about 1×1012 target sequence copiesper mm2. In some instances, the surface density of target sequencecopies may be at least 100, at least 500, at least 1,000, at least5,000, at least 10,000, at least 15,000, at least 20,000, at least25,000, at least 30,000, at least 35,000, at least 40,000, at least45,000, at least 50,000, at least 55,000, at least 60,000, at least65,000, at least 70,000, at least 75,000, at least 80,000, at least85,000, at least 90,000, at least 95,000, at least 100,000, at least150,000, at least 200,000, at least 250,000, at least 300,000, at least350,000, at least 400,000, at least 450,000, at least 500,000, at least550,000, at least 600,000, at least 650,000, at least 700,000, at least750,000, at least 800,000, at least 850,000, at least 900,000, at least950,000, at least 1,000,000, at least 5,000,000, at least 1×107, atleast 5×107, at least 1×108, at least 5×108, at least 1×109, at least5×109, at least 1×1010, at least 5×1010, at least 1×1011, at least5×1011, or at least 1×1012 of clonally amplified target sequencemolecules per mm2. In some instances, the surface density of targetsequence copies may be at most 1×1012, at most 5×1011, at most 1×1011,at most 5×1010, at most 1×1010, at most 5×109, at most 1×109, at most5×108, at most 1×108, at most 5×107, at most 1×107, at most 5,000,000,at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, atmost 800,000, at most 750,000, at most 700,000, at most 650,000, at most600,000, at most 550,000, at most 500,000, at most 450,000, at most400,000, at most 350,000, at most 300,000, at most 250,000, at most200,000, at most 150,000, at most 100,000, at most 95,000, at most90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000,at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000,at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most1,000, at most 500, or at most 100 target sequence copies per mm2. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of target sequence copiesmay range from about 1,000 target sequence copies per mm2 to about65,000 target sequence copies mm2. Those of skill in the art willrecognize that the surface density of target sequence copies may haveany value within this range, e.g., about 49,600 target sequence copiesper mm2.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification bufferformulations may result in a surface density of clonally-amplifiedtarget (or sample) oligonucleotide molecules (or clusters) ranging fromabout from about 100 molecules per mm2 to about 1×1012 colonies per mm2.In some instances, the surface density of clonally-amplified moleculesmay be at least 100, at least 500, at least 1,000, at least 5,000, atleast 10,000, at least 15,000, at least 20,000, at least 25,000, atleast 30,000, at least 35,000, at least 40,000, at least 45,000, atleast 50,000, at least 55,000, at least 60,000, at least 65,000, atleast 70,000, at least 75,000, at least 80,000, at least 85,000, atleast 90,000, at least 95,000, at least 100,000, at least 150,000, atleast 200,000, at least 250,000, at least 300,000, at least 350,000, atleast 400,000, at least 450,000, at least 500,000, at least 550,000, atleast 600,000, at least 650,000, at least 700,000, at least 750,000, atleast 800,000, at least 850,000, at least 900,000, at least 950,000, atleast 1,000,000, at least 5,000,000, at least 1×107, at least 5×107, atleast 1×108, at least 5×108, at least 1×109, at least 5×109, at least1×1010, at least 5×1010, at least 1×1011, at least 5×1011, or at least1×1012 molecules per mm2. In some instances, the surface density ofclonally-amplified molecules may be at most 1×1012, at most 5×1011, atmost 1×1011, at most 5×1010, at most 1×1010, at most 5×109, at most1×109, at most 5×108, at most 1×108, at most 5×107, at most 1×107, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm2. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedmolecules may range from about 5,000 molecules per mm2 to about 50,000molecules per mm2. Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 molecules per mm2.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification bufferformulations may result in a surface density of clonally-amplifiedtarget (or sample) oligonucleotide molecules (or clusters) ranging fromabout from about 100 molecules per mm2 to about 1×1012 colonies per mm2.In some instances, the surface density of clonally-amplified moleculesmay be at least 100, at least 500, at least 1,000, at least 5,000, atleast 10,000, at least 15,000, at least 20,000, at least 25,000, atleast 30,000, at least 35,000, at least 40,000, at least 45,000, atleast 50,000, at least 55,000, at least 60,000, at least 65,000, atleast 70,000, at least 75,000, at least 80,000, at least 85,000, atleast 90,000, at least 95,000, at least 100,000, at least 150,000, atleast 200,000, at least 250,000, at least 300,000, at least 350,000, atleast 400,000, at least 450,000, at least 500,000, at least 550,000, atleast 600,000, at least 650,000, at least 700,000, at least 750,000, atleast 800,000, at least 850,000, at least 900,000, at least 950,000, atleast 1,000,000, at least 5,000,000, at least 1×107, at least 5×107, atleast 1×108, at least 5×108, at least 1×109, at least 5×109, at least1×1010, at least 5×1010, at least 1×1011, at least 5×1011, or at least1×1012 molecules per mm2. In some instances, the surface density ofclonally-amplified molecules may be at most 1×1012, at most 5×1011, atmost 1×1011, at most 5×1010, at most 1×1010, at most 5×109, at most1×109, at most 5×108, at most 1×108, at most 5×107, at most 1×107, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm2. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedmolecules may range from about 5,000 molecules per mm2 to about 50,000molecules per mm2. Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 molecules per mm2.

In some instances, the use of the disclosed low non-specific bindingsupports alone or in combination with optimized amplification bufferformulations may result in a surface density of clonally-amplifiedtarget (or sample) oligonucleotide colonies (or clusters) ranging fromabout from about 100 colonies per mm2 to about 1×1012 colonies per mm2.In some instances, the surface density of clonally-amplified coloniesmay be at least 100, at least 500, at least 1,000, at least 5,000, atleast 10,000, at least 15,000, at least 20,000, at least 25,000, atleast 30,000, at least 35,000, at least 40,000, at least 45,000, atleast 50,000, at least 55,000, at least 60,000, at least 65,000, atleast 70,000, at least 75,000, at least 80,000, at least 85,000, atleast 90,000, at least 95,000, at least 100,000, at least 150,000, atleast 200,000, at least 250,000, at least 300,000, at least 350,000, atleast 400,000, at least 450,000, at least 500,000, at least 550,000, atleast 600,000, at least 650,000, at least 700,000, at least 750,000, atleast 800,000, at least 850,000, at least 900,000, at least 950,000, atleast 1,000,000, at least 5,000,000, at least 1×107, at least 5×107, atleast 1×108, at least 5×108, at least 1×109, at least 5×109, at least1×1010, at least 5×1010, at least 1×1011, at least 5×1011, or at least1×1012 colonies per mm2. In some instances, the surface density ofclonally-amplified colonies may be at most 1×1012, at most 5×1011, atmost 1×1011, at most 5×1010, at most 1×1010, at most 5×109, at most1×109, at most 5×108, at most 1×108, at most 5×107, at most 1×107, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 colonies per mm2. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedcolonies may range from about 5,000 colonies per mm2 to about 50,000colonies per mm2. Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 colonies per mm2.

In some cases the use of the disclosed low non-specific binding supportsalone or in combination with optimized amplification reactionformulations may yield signal from the amplified and labeled nucleicacid populations (e.g., a fluorescence signal) that has a coefficient ofvariance of no greater than 50%, such as 50%, 40%, 30%, 20%, 15%, 10%,5%, or less than 5%.

In some cases, the support surfaces and methods as disclosed hereinallow amplification at elevated extension temperatures, such as at 15 C,20 C, 25 C, 30 C, 40 C, or greater, or for example at about 21 C or 23C.

In some cases, the use of the support surfaces and methods as disclosedherein enable simplified amplification reactions. For example, in somecases amplification reactions are performed using no more than 1, 2, 3,4, or 5 discrete reagents.

In some cases, the use of the support surfaces and methods as disclosedherein enable the use of simplified temperature profiles duringamplification, such that reactions are executed at temperatures rangingfrom a low temperature of 15 C, 20 C, 25 C, 30 C, or 40 C, to a hightemperature of 40 C, 45 C, 50 C, 60 C, 65 C, 70 C, 75 C, 80 C, orgreater than 80 C, for example, such as a range of 20 C to 65 C.

Amplification reactions are also improved such that lower amounts oftemplate (e.g., target or sample molecules) are sufficient to lead todiscernable signals on a surface, such as 1 pM, 2 pM, 5 pM, 10 pM, 15pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1,000 pM,2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM,9,000 pM, 10,000 pM or greater than 10,000 pM of a sample, such as 500nM. In exemplary embodiments, inputs of about 100 pM are sufficient togenerate signals for reliable signal determination.

The disclosed solid-phase nucleic acid amplification reactionformulations and low non-specific binding supports may be used in any ofa variety of nucleic acid analysis applications, e.g., nucleic acid basediscrimination, nucleic acid base classification, nucleic acid basecalling, nucleic acid detection applications, nucleic acid sequencingapplications, and nucleic acid-based (genetic and genomic) diagnosticapplications. In many of these applications, fluorescence imagingtechniques may be used to monitor hybridization, amplification, and/orsequencing reactions performed on the low-binding supports.

Fluorescence imaging may be performed using any of a variety offluorophores, fluorescence imaging techniques, and fluorescence imaginginstruments known to those of skill in the art. Examples of suitablefluorescence dyes that may be used (e.g., by conjugation to nucleotides,oligonucleotides, or proteins) include, but are not limited to,fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof,including the cyanine derivatives Cyanine dye-3 (Cy3), Cyanine dye-5(Cy5), Cyanine dye-7 (Cy7), etc. Examples of fluorescence imagingtechniques that may be used include, but are not limited to,fluorescence microscopy imaging, fluorescence confocal imaging,two-photon fluorescence, and the like. Examples of fluorescence imaginginstruments that may be used include, but are not limited to,fluorescence microscopes equipped with an image sensor or camera,confocal fluorescence microscopes, two-photon fluorescence microscopes,or custom instruments that comprise a suitable selection of lightsources, lenses, mirrors, prisms, dichroic reflectors, apertures, andimage sensors or cameras, etc. A non-limiting example of a fluorescencemicroscope equipped for acquiring images of the disclosed low-bindingsupport surfaces and clonally-amplified colonies (or clusters) of targetnucleic acid sequences hybridized thereon is the Olympus IX83 invertedfluorescence microscope equipped with) 20×, 0.75 NA, a 532 nm lightsource, a bandpass and dichroic mirror filter set optimized for 532 nmlong-pass excitation and Cy3 fluorescence emission filter, a Semrock 532nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) where theexcitation light intensity is adjusted to avoid signal saturation.Often, the support surface may be immersed in a buffer (e.g., 25 mMACES, pH 7.4 buffer) while the image is acquired.

In some instances, the performance of nucleic acid hybridization and/oramplification reactions using the disclosed reaction formulations andlow non-specific binding supports may be assessed using fluorescenceimaging techniques, where the contrast-to-noise ratio (CNR) of theimages provides a key metric in assessing amplification specificity andnon-specific binding on the support. CNR is commonly defined as:CNR=(Signal−Background)/Noise. The background term is commonly taken tobe the signal measured for the interstitial regions surrounding aparticular feature (diffraction limited spot, DLS) in a specified regionof interest (ROI). While signal-to-noise ratio (SNR) is often consideredto be a benchmark of overall signal quality, it can be shown thatimproved CNR can provide a significant advantage over SNR as a benchmarkfor signal quality in applications that require rapid image capture(e.g., sequencing applications for which cycle times must be minimized),as shown in the example below. The surfaces of the instant disclosureare also provided in co-pending International Application Serial No.PCT/US2019/061556, which is hereby incorporated by reference in itsentirety.

In most ensemble-based sequencing approaches, the background term istypically measured as the signal associated with ‘interstitial’ regions.In addition to “interstitial” background (B_(inter)), “intrastitial”background (B_(intra)) exists within the region occupied by an amplifiedDNA colony. The combination of these two background signals dictates theachievable CNR, and subsequently directly impacts the optical instrumentrequirements, architecture costs, reagent costs, run-times, cost/genome,and ultimately the accuracy and data quality for cyclic array-basedsequencing applications. The B_(inter) background signal arises from avariety of sources; a few examples include auto-fluorescence fromconsumable flow cells, non-specific adsorption of detection moleculesthat yield spurious fluorescence signals that may obscure the signalfrom the ROI, the presence of non-specific DNA amplification products(e.g., those arising from primer dimers). In typical next generationsequencing (NGS) applications, this background signal in the currentfield-of-view (FOV) is averaged over time and subtracted. The signalarising from individual DNA colonies (i.e., (S)−B_(inter) in the FOV)yields a discernable feature that can be classified. In some instances,the intrastitial background (B_(intra)) can contribute a confoundingfluorescence signal that is not specific to the target of interest, butis present in the same ROI thus making it far more difficult to averageand subtract.

As will be demonstrated in the examples below, the implementation ofnucleic acid amplification on the low-binding substrates of the presentdisclosure may decrease the B_(inter) background signal by reducingnon-specific binding, may lead to improvements in specific nucleic acidamplification, and may lead to a decrease in non-specific amplificationthat can impact the background signal arising from both the interstitialand intrastitial regions. In some instances, the disclosed low-bindingsupport surfaces, optionally used in combination with the disclosedhybridization and/or amplification reaction formulations, may lead toimprovements in CNR by a factor of 2, 5, 10, 100, or 1000-fold overthose achieved using conventional supports and hybridization,amplification, and/or sequencing protocols. Although described here inthe context of using fluorescence imaging as the read-out or detectionmode, the same principles apply to the use of the disclosed lownon-specific binding supports and nucleic acid hybridization andamplification formulations for other detection modes as well, includingboth optical and non-optical detection modes.

The disclosed low-binding supports, optionally used in combination withthe disclosed hybridization and/or amplification protocols, yieldsolid-phase reactions that exhibit: (i) negligible non-specific bindingof protein and other reaction components (thus minimizing substratebackground), (ii) negligible non-specific nucleic acid amplificationproduct, and (iii) provide tunable nucleic acid amplification reactions.

Methods for Capturing and Analyzing DNA. The present disclosure providesmethods for analyzing nucleic acids in a manner that is cellularly orspatially addressable, the method comprising: (a) providing a supportcomprising a low non-specific binding coating to which a plurality ofcapture oligonucleotides and a plurality of circularizationoligonucleotides are immobilized (e.g., FIG. 2), wherein the pluralityof capture oligonucleotides comprise (i) a target capture region thathybridizes to at least a portion of a target nucleic acid molecule, (ii)a universal sequence region comprising a spatial barcode sequence, (iii)a circularization anchor sequence, and (iv) a cleavable region, whereinthe plurality of circularization oligonucleotides comprise (i) ahomopolymer region, (ii) a universal sequence region comprising asequencing primer binding sequence and (iii) a circularization anchorbinding sequence, and wherein the low non-specific binding coatingcomprises at least one hydrophilic polymer coating having a watercontact angle of no more than 45 degrees.

In some embodiments, the low non-specific binding coating in step (a)exhibits low background fluorescence signals or high contrast to noise(CNR) ratios relative to known surfaces in the art. In some embodiments,the low non-specific binding coating exhibits a level of non-specificCy3 dye absorption of less than about 0.25 molecules/μm², where no morethan 5% of the target nucleic acid is associated with the surfacecoating without hybridizing to an immobilized capture oligonucleotide.In some embodiments, a fluorescence image of the surface coating havinga plurality of clonally-amplified clusters of nucleic acid exhibits acontrast-to-noise ratio (CNR) of at least 20, or at least 50, or highercontrast-to-noise ratios (CNR), when using a fluorescence imaging systemunder non-signal saturating conditions.

In some embodiments, the immobilized capture oligonucleotide in step (a)can include any combination of: (i) a target capture region thathybridizes to at least a portion of a target nucleic acid molecule, (ii)a universal sequence region comprising a spatial barcode sequence, (iii)a circularization anchor sequence that binds a portion of thecircularization oligonucleotide, and/or (iv) a cleavable region.

In some embodiments, the target capture region of the immobilizedcapture oligonucleotides in step (a) comprise a target-specific sequenceor a random sequence.

In some embodiments, the immobilized circularization oligonucleotides instep (a) can include any combination of: (i) a homopolymer region, (ii)a universal sequence region comprising a sequencing primer bindingsequence and/or (iii) a circularization anchor binding sequence thatbinds the circularization anchor sequence of the captureoligonucleotide.

The method for analyzing nucleic acids further comprises the step: (b)contacting the low non-specific binding coating with a cellularbiological sample in the presence of a high efficiency hybridizationbuffer under a condition suitable to promote migration of the targetnucleic acid molecule from the cellular biological sample to one of theimmobilized capture oligonucleotides thereby forming an immobilizedtarget nucleic acid duplex, wherein the target nucleic acid molecule isimmobilized to the low non-specific binding coating in a manner thatpreserves spatial location information of the target nucleic acidmolecule in the cellular biological sample, wherein the target nucleicacid comprises DNA or RNA (e.g., FIG. 7).

In some embodiments, the cellular biological sample in step (b)comprises a cellular biological sample that is fresh, frozen, freshfrozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE).

In some embodiments, the cellular biological sample in step (b) issubjected to a permeabilizing reaction to promote migration of thecellular nucleic acid molecules (e.g., DNA and/or RNA), including thetarget nucleic acid molecule, from the cellular biological sample to oneof the immobilized capture oligonucleotides.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the high efficiency highefficiency hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the high efficiency highefficiency hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe high efficiency high efficiency hybridization buffer. In someembodiments, the high efficiency hybridization buffer further comprisesbetaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

The method for analyzing nucleic acids further comprises the step: (c)conducting a primer extension reaction on the immobilized nucleic acidduplex using the hybridized target nucleic acid molecule as a templatethereby forming an immobilized target extension product. In someembodiments, the primer extension reaction comprises contacting theimmobilized nucleic acid duplex with a plurality of nucleotides and apolymerase. In some embodiments, the polymerase comprises an E. coli DNApolymerase I, Klenow fragment of E. coli DNA polymerase I, T7 DNApolymerase, or T4 DNA polymerase.

In some embodiments, the primer extension reaction of step (c) can be areverse transcription reaction which comprises (i) a reversetranscriptase enzyme, (ii) a plurality of nucleotides, and (iii) aplurality of reverse transcriptase primers. In some embodiments, thereverse transcription reaction of step (a) comprises a plurality ofnucleotides and an enzyme having reverse transcription activity,including reverse transcriptase enzymes from AMV (avian myeloblastosisvirus), M-MLV (moloney murine leukemia virus), or HIV (humanimmunodeficiency virus). In some embodiments, the reverse transcriptasecan be a commercially-available enzyme, including MultiScribe™,ThermoScript™, or ArrayScript™. In some embodiments, the reversetranscriptase enzyme comprises Superscript I, II, III, or IV enzymes. Insome embodiments, the reverse transcription reaction can include anRNase inhibitor.

The method for analyzing nucleic acids further comprises the step: (d)conducting a non-template tailing reaction on the immobilized targetextension product under conditions suitable for appending a homopolymertail to the immobilized target extension product thereby forming animmobilized tailed target extension product (e.g., FIG. 27). In someembodiments, the non-template tailing reaction comprises contacting theimmobilized target extension product with a plurality of nucleotides anda polymerase where the polymerase is a Taq polymerase, Tfi DNApolymerase, 3′ exonuclease minus-large (Klenow) fragment, or 3′exonuclease minus-T4 polymerase.

The method for analyzing nucleic acids further comprises the step: (e)cleaving the immobilized tailed target extension product to release theimmobilized tailed target extension product from the low binding coatingthereby forming a soluble tailed target extension product. In someembodiments, the cleavable region can be cleaved with an enzyme, achemical compound, light or heat.

The method for analyzing nucleic acids further comprises the step: (f)binding the soluble tailed target extension product to one of theimmobilized circularization oligonucleotides under a condition suitableto hybridize the appended homopolymer tail of the soluble tailed targetextension product to the homopolymer region of the immobilizedcircularization oligonucleotide, and suitable to hybridize thecircularization anchor sequence of the soluble tailed target extensionproduct to the circularization anchor binding sequence of theimmobilized circularization oligonucleotide thereby forming an opencircular target extension product with a gap and/or nick, such that theimmobilized circularization oligonucleotide serves as a splint moleculeto promote circularization of the soluble tailed target extensionproduct (e.g., FIG. 27).

The method for analyzing nucleic acids further comprises the step: (g)closing the gap (if present) by conducting a gap-filling primerextension reaction and closing the nick (if present) by conducting aligation reaction on the open circular target extension product therebyforming a covalently closed circular target extension product which ishybridized to the immobilized circularization oligonucleotide, whereinthe immobilized circularization oligonucleotide includes a homopolymerregion with a 3′ extendible end (e.g., FIG. 27).

In some embodiments, the forming the covalently closed circular targetextension product of step (g) comprises a polymerase-mediatedgap-filling reaction, an enzymatic ligation reaction, or apolymerase-mediated gap-filling reaction and enzymatic ligationreaction. In some embodiments, the polymerase-mediate gap-fillingreaction comprises contacting the open circular target molecule with aDNA polymerase and a plurality of nucleotides, where the DNA polymerasecomprises E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T7 DNA polymerase, or T4 DNA polymerase. In someembodiments, the enzymatic ligation reaction comprises use of a ligaseenzyme, including a T3, T4, T7 or Taq DNA ligase enzyme. In someembodiments, the forming the covalently closed circular target moleculecomprises contacting the open circular target molecule with a CircLigaseor CircLigase II enzyme.

The method for analyzing nucleic acids further comprises the step: (h)conducting a rolling circle amplification reaction using the 3′extendible end of the homopolymer region of the immobilizedcircularization oligonucleotide under a condition suitable to form animmobilized nucleic acid concatemer molecule having tandem repeatregions comprising the sequencing primer binding sequence, the targetsequence, and the spatial barcode sequence (e.g., FIG. 27).

In some embodiments, the rolling circle amplification reaction of step(h) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(h) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(h) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C., or from room temperature to about65° C.

In some embodiments, the rolling circle amplification reaction of step(h) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(h) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiment, the rolling circle amplification reaction can befollowed by a flexing amplification reaction instead of a multipledisplacement amplification (MDA) reaction. In some embodiments, theflexing amplification reaction comprises: (a) forming a nucleic acidrelaxant reaction mixture by contacting the nucleic acid concatemer withone or a combination of two or more compounds selected from a groupconsisting of formamide, acetonitrile, ethanol, guanidine hydrochloride,urea, potassium iodide and/or polyamines, to generate a relaxed nucleicacid concatemer, wherein the forming a nucleic acid relaxant reactionmixture is conducted with a temperature ramp-up, a relaxant incubationtemperature, and a temperature ramp-down; (b) washing the relaxedconcatemer; (c) forming a flexing amplification reaction mixture bycontacting the relaxed concatemer with a strand-displacing DNApolymerase, a plurality of nucleotides, a catalytic divalent cation, (inthe absence of added amplification primers), to generate double-strandedconcatemers, wherein the forming a flexing amplification reactionmixture is conducted with a temperature ramp-up, a flexing incubationtemperature, and a temperature ramp-down; (d) washing thedouble-stranded concatemer; and (e) repeating steps (a)-(d) at leastonce.

Methods of Capturing and Analyzing RNA. Provided herein are methods foranalyzing nucleic acids (e.g., RNA), comprising: (a) providing a supportcomprising a low non-specific binding coating to which a plurality ofcapture oligonucleotides are immobilized (e.g., FIGS. 4 and 28), whereinthe plurality of capture oligonucleotides comprise (i) a target captureregion that hybridizes to at least a portion of a target nucleic acidmolecule, (ii) a universal sequence region comprising a spatial barcodesequence and optionally a sample barcode sequence, and (iii) a cleavableregion, wherein low non-specific binding coating comprises at least onehydrophilic polymer coating having a water contact angle of no more than45 degrees. In some embodiments, the target capture region comprises ahomopolymer region having a poly-T sequence.

In some embodiments, the low non-specific binding coating in step (a)exhibits low background fluorescence signals or high contrast to noise(CNR) ratios relative to known surfaces in the art. In some embodiments,the low non-specific binding coating exhibits a level of non-specificCy3 dye absorption of less than about 0.25 molecules/μm², where no morethan 5% of the target nucleic acid is associated with the surfacecoating without hybridizing to an immobilized capture oligonucleotide.In some embodiments, a fluorescence image of the surface coating havinga plurality of clonally-amplified clusters of nucleic acid exhibits acontrast-to-noise ratio (CNR) of at least 20, or at least 50, or highercontrast-to-noise ratios (CNR), when using a fluorescence imaging systemunder non-signal saturating conditions.

The method for analyzing nucleic acids further comprises the step: (b)contacting the low non-specific binding coating with a cellularbiological sample in the presence of a high efficiency hybridizationbuffer under a condition suitable to promote migration of the targetnucleic acid molecule from the cellular biological sample to one of theimmobilized capture oligonucleotides thereby forming an immobilizedtarget nucleic acid duplex, wherein the target nucleic acid molecule isimmobilized to the low non-specific binding coating in a manner thatpreserves spatial location information of the target nucleic acidmolecule in the cellular biological sample, wherein the target nucleicacid comprises a poly-A RNA molecule. In some embodiments, the targetcapture region having a poly-T sequence can hybridize to poly-A RNA(e.g., FIG. 28).

In some embodiments, the cellular biological sample in step (b)comprises a cellular biological sample that is fresh, frozen, freshfrozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE).

In some embodiments, the cellular biological sample in step (b) issubjected to a permeabilizing reaction to promote migration of thecellular nucleic acid molecules (e.g., DNA and/or RNA), including thetarget nucleic acid molecule, from the cellular biological sample to oneof the immobilized capture oligonucleotides.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the high efficiency highefficiency hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the high efficiency highefficiency hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe high efficiency high efficiency hybridization buffer. In someembodiments, the high efficiency hybridization buffer further comprisesbetaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

The method for analyzing nucleic acids further comprises the step: (c)conducting a reverse transcription reaction on the immobilized nucleicacid duplex using the hybridized target nucleic acid molecule as atemplate thereby forming an immobilized target extension product (e.g.,cDNA) (e.g., FIG. 28).

In some embodiments, the reverse transcription reaction of step (c)comprises (i) a reverse transcriptase enzyme, (ii) a plurality ofnucleotides, and (iii) a plurality of reverse transcriptase primers. Insome embodiments, the reverse transcription reaction of step (a)comprises a plurality of nucleotides and an enzyme having reversetranscription activity, including reverse transcriptase enzymes from AMV(avian myeloblastosis virus), M-MLV (moloney murine leukemia virus), orHIV (human immunodeficiency virus). In some embodiments, the reversetranscriptase can be a commercially-available enzyme, including MultiScribe™, ThermoScript™, or ArrayScript™. In some embodiments, thereverse transcriptase enzyme comprises Superscript I, II, III, or IVenzymes. In some embodiments, the reverse transcription reaction caninclude an RNase inhibitor.

In some embodiments, the method for analyzing nucleic acids (e.g., RNA)further comprises: (d) appending a nucleic acid adaptor to thenon-immobilized end of the immobilized target extension product therebygenerating an adaptor-appended immobilized double-stranded targetextension product (FIG. 28). The nucleic acid adaptor can besingle-stranded or double-stranded. The nucleic acid adaptor can beappended using an RNA ligase or DNA ligase. Single-stranded adaptors canbe appended to the 3′ end of one strand of the immobilized targetextension product using T4 RNA ligase, KOD ligase, Circligase, orSplintR ligase. Double-stranded adaptors can be appended to thenon-immobilized end of the immobilized target extension product using T4DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9degrees N) DNA ligase, Ampligase, or SplintR ligase. Theadaptor-appended immobilized double-stranded target extension productcomprises the immobilized capture oligonucleotide (extended via reversetranscription and appended with an adaptor) which is hybridized to thetarget nucleic acid molecule. In some embodiments the adaptor-appendedimmobilized double-stranded target extension product is subjected to acondition that dissociates/removes or degrades the target nucleic acidmolecule so that the adaptor-appended immobilized single-stranded targetextension product remains attached to the surface.

The method for analyzing nucleic acid may further comprises the step:(e) contacting the adaptor-appended immobilized single-stranded targetextension product with plurality of soluble circularizationoligonucleotides to form a target-circularization duplex, wherein thesoluble circularization oligonucleotides each comprise (i) an adaptorbinding region, (ii) a homopolymer region (iii) an anchor region, and(iv) an anchor moiety, wherein the homopolymer region comprises a poly-Tsequence that can hybridize to the poly-A region of the target nucleicacid molecule, wherein the contacting is conducted under a conditionsuitable to immobilize at least one of the soluble circularizationoligonucleotides to the low non-specific binding coating in closeproximity to the adaptor-appended immobilized single-stranded targetextension product (e.g., FIG. 28).

In some embodiments, the adaptor binding region includes a sequencingprimer binding region. In some embodiments, the adaptor binding regioninclude an amplification primer binding region. In some embodiments, thehomopolymer region comprises a polynucleotide sequence selected from agroup consisting of poly-T, poly-dT, poly-A, poly-dA, poly-C, poly-dC,poly-G and poly-dG. In some embodiments, the homopolymer regioncomprises a poly-T or poly-dT sequence. In some embodiments, the anchormoiety can attach to the surface thereby generating an immobilizedcircularization oligonucleotide. The adaptor binding region of theimmobilized circularization oligonucleotide can hybridize to theappended adaptor sequence of the adaptor-appended immobilizedsingle-stranded target extension product. The homopolymer region of theimmobilized circularization oligonucleotide can hybridize to thehomopolymer region (e.g., poly-A) of the adaptor-appended immobilizedsingle-stranded target extension product.

The method for analyzing nucleic acids may further comprises the step:(f) cleaving the cleavable region of the target-circularization duplexto release the immobilized end from the low non-specific binding coatingto generate a released target extension product, wherein the appendedadaptor region of the released target extension product remainshybridized to the adaptor-binding region of the immobilizedcircularization oligonucleotide, and homopolymer region of the releasedtarget extension product can re-hybridize with the homopolymer region ofthe immobilized circularization oligonucleotide thereby forming an opencircular target-circularization duplex with a gap and/or a nick, suchthat the immobilized circularization oligonucleotide serves as a splintmolecule to promote circularization of the released target extensionproduct (e.g., FIG. 28). In some embodiments, the cleavable region canbe cleaved with an enzyme, a chemical compound, light or heat. In someembodiments, the appended adaptor region of the released targetextension product remains hybridized to the adaptor-appended immobilizedsingle-stranded target extension product. In some embodiments, thehomopolymer region of the released target extension product canre-hybridize with the homopolymer region of the immobilizedcircularization oligonucleotide thereby forming an open circularizedadaptor-appended target extension product with a gap or a nick. Theimmobilized circularization oligonucleotide can serve as a splintmolecule to promote circularization of the released target extensionproduct, as the homopolymer region and the adaptor binding region of theimmobilized circularization oligonucleotide can hybridize to the ends ofthe released target extension product.

The method for analyzing nucleic acids may further comprises the step:(g) closing the gap (if present) by conducting a gap-filling primerextension reaction and closing the nick (if present) by conducting aligation reaction on the open circular target-circularization duplexthereby forming a covalently closed circular target extension productwhich is hybridized to the immobilized circularization oligonucleotide,wherein the immobilized circularization oligonucleotide includes anadaptor-binding region with a 3′ extendible end (e.g., FIG. 28).

In some embodiments, the forming the covalently closed circular targetextension product of step (g) comprises a polymerase-mediatedgap-filling reaction, an enzymatic ligation reaction, or apolymerase-mediated gap-filling reaction and enzymatic ligationreaction. In some embodiments, the polymerase-mediate gap-fillingreaction comprises contacting the open circular target molecule with aDNA polymerase and a plurality of nucleotides, where the DNA polymerasecomprises E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T7 DNA polymerase, or T4 DNA polymerase. In someembodiments, the enzymatic ligation reaction comprises use of a ligaseenzyme, including a T3, T4, T7 or Taq DNA ligase enzyme. In someembodiments, the forming the covalently closed circular target moleculecomprises contacting the open circular target molecule with a CircLigaseor CircLigase II enzyme.

The method for analyzing nucleic acids may further comprises the step:(h) conducting a rolling circle amplification reaction by extending the3′ extendible end of the adaptor binding region of the immobilizedcircularization oligonucleotide under a condition suitable to form animmobilized nucleic acid concatemer molecule having tandem repeatregions comprising the sequencing primer binding sequence, the targetsequence, and the spatial barcode sequence (e.g., FIG. 28).

In some embodiments, the rolling circle amplification reaction of step(h) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(h) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(h) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C., or from room temperature to about65° C.

In some embodiments, the rolling circle amplification reaction of step(h) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(h) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiment, the rolling circle amplification reaction can befollowed by a flexing amplification reaction instead of a multipledisplacement amplification (MDA) reaction. In some embodiments, theflexing amplification reaction comprises: (a) forming a nucleic acidrelaxant reaction mixture by contacting the nucleic acid concatemer withone or a combination of two or more compounds selected from a groupconsisting of formamide, acetonitrile, ethanol, guanidine hydrochloride,urea, potassium iodide and/or polyamines, to generate a relaxed nucleicacid concatemer, wherein the forming a nucleic acid relaxant reactionmixture is conducted with a temperature ramp-up, a relaxant incubationtemperature, and a temperature ramp-down; (b) washing the relaxedconcatemer; (c) forming a flexing amplification reaction mixture bycontacting the relaxed concatemer with a strand-displacing DNApolymerase, a plurality of nucleotides, a catalytic divalent cation, (inthe absence of added amplification primers), to generate double-strandedconcatemers, wherein the forming a flexing amplification reactionmixture is conducted with a temperature ramp-up, a flexing incubationtemperature, and a temperature ramp-down; (d) washing thedouble-stranded concatemer; and (e) repeating steps (a)-(d) at leastonce.

Methods and Compositions for Nucleic Acid Determination. Provided hereinare methods for analyzing nucleic acid comprising determining thesequence of the target nucleic acid (e.g., immobilized concatemer)referred to herein. The sequencing may be targeted sequencing. Thesequencing may be whole genome sequencing. Whole genome sequencing maycomprise massive parallel sequencing (“next generation sequencing” or“second generation sequencing”). In some embodiments, the sequencing isperformed by ligation. In some embodiments, the sequencing comprises thesequential monitoring of incorporation of labeled nucleotides in growingpolynucleotide molecule. Sequencing may be performed by massivelyparallel array sequencing or single molecule sequencing.

The method for analyzing nucleic acids further comprises the step: (i)sequencing at least a portion of the immobilized nucleic acidconcatemer, including sequencing the target sequence and the spatialbarcode sequence, to determine the spatial location of the targetnucleic acid in the cellular biological sample.

In some embodiments, the sequencing of step (i) comprises sequencing atleast a portion of the nucleic acid concatemers using an optical imagingsystem comprising a field-of-view (FOV) greater than 1.0 mm².

In some embodiments, the sequencing of step (i) includes placing thecellular biological sample in a flow cell having walls (e.g., top orfirst wall, and bottom or second wall) and a gap in-between, where thegap can be filled with a fluid, where the flow cell is positioned in afluorescence optical imaging system. The cellular biological sample hasa thickness that may require using the imaging system to focusseparately on the first and second surfaces of the flow cell, when usinga traditional imaging system. For improved imaging of the sequencingreaction of the nucleic acids from the cellular biological sample, theflow cell can be positioned in a high performance fluorescence imagingsystem, which comprises two or more tube lenses which are designed toprovide optimal imaging performance for the first and second surfaces ofthe flow cell at two or more fluorescence wavelengths. In someembodiments, the high-performance imaging system further comprises afocusing mechanism configured to refocus the optical system betweenacquiring images of the first and second surfaces of the flow cell. Insome embodiments, the high performance imaging system is configured toimage two or more fields-of-view on at least one of the first flow cellsurface or the second flow cell surface.

In some embodiments, the sequencing of step (i) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker.

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

In some embodiments, the multivalent molecule comprises: (a) a core, and(b) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms. In some embodiments, the spacer is attached to thelinker. In some embodiments, the linker is attached to the nucleotideunit. In some embodiments, the nucleotide unit comprises a base, sugarand at least one phosphate group, and wherein the linker is attached tothe nucleotide unit through the base. In some embodiments, the linkercomprises an aliphatic chain or an oligo ethylene glycol chain whereboth linker chains having 2-6 subunits and optionally the linkerincludes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (i) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (1) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (i) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

The sequencing method can include contacting a target nucleic acid ormultiple target nucleic acids, comprising multiple linked or unlinkedcopies of a target sequence, with the multivalent binding compositionsdescribed herein. Contacting said target nucleic acid, or multipletarget nucleic acids comprising multiple linked or unlinked copies of atarget sequence, with one or more polymer-nucleotide conjugates mayprovide a substantially increased local concentration of the correctnucleotide being interrogated in a given sequencing cycle, thussuppressing signals from improper incorporations or phased nucleic acidchains (i.e., those elongating nucleic acid chains which have had one ormore skipped cycles).

Provided herein are methods of obtaining nucleic acid sequenceinformation comprising contacting a target nucleic acid, or multipletarget nucleic acids, with one or more polymer-nucleotide conjugates. Insome embodiments, the target nucleic acid or multiple target nucleicacids comprise multiple linked or unlinked copies of a target sequence.In some embodiments, the method results in a reduction in the error rateof sequencing as indicated by reduction in the misidentification ofbases, the reporting of nonexistent bases, or the failure to reportcorrect bases. In some embodiments, said reduction in the error orate ofsequencing may comprise a reduction of 5%, 10%, 15%, 20% 25%, 50%, 75%,100%, 150%, 200%, or more compared to the error rate observed usingmonovalent ligands, including free nucleotides, labeled freenucleotides, protein or peptide bound nucleotides, or labeled protein orpeptide bound nucleotides. In some embodiments, the method results in anincrease in average read length of 5%, 10%, 15%, 20% 25%, 50%, 75%,100%, 150%, 200%, 300%, or more compared to the average read lengthobserved using monovalent ligands, including free nucleotides, labeledfree nucleotides, protein or peptide bound nucleotides, or labeledprotein or peptide bound nucleotides. In some embodiments, the methodresults in an increase in average read length of 10, 20, 25, 30, 50, 75,100, 125, 150, 200, 250, 300, 350, 400, 500 nucleotides, or morecompared to the average read length observed using monovalent ligands,including free nucleotides, labeled free nucleotides, protein or peptidebound nucleotides, or labeled protein or peptide bound nucleotides.

The use of the polymer-nucleotide conjugates for sequencing can shortensthe total time of a sequencing reaction or sequencing run. Thesequencing reaction cycle comprising the contacting, detecting, andincorporating steps is performed in a total time ranging from about 5minutes to about 60 minutes. In some embodiments, the sequencingreaction cycle is performed in at least 5 minutes, at least 10 minutes,at least 20 minutes, at least 30 minutes, at least 40 minutes, at least50 minutes, or at least 60 minutes. In some embodiments, the sequencingreaction cycle is performed in at most 60 minutes, at most 50 minutes,at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10minutes, or at most 5 minutes. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some embodiments thesequencing reaction cycle may be performed in a total time ranging fromabout 10 minutes to about 30 minutes. Those of skill in the art willrecognize that the sequencing cycle time may have any value within thisrange, e.g., about 16 minutes.

The use of the polymer-nucleotide conjugates for sequencing provides anmore accuracy base readout. The disclosed compositions and methods fornucleic acid sequencing will provide an average Q-score for base-callingaccuracy over a sequencing run that ranges from about 20 to about 50. Insome embodiments, the average Q-score is at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50. Thoseof skill in the art will recognize that the average Q-score may have anyvalue within this range, e.g., about 32. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 30 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 35for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 40 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 45for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

The present disclosure relates to polymer-nucleotide conjugates eachhaving a plurality of nucleotides conjugated to a particle or core(e.g., a polymer, branched polymer, dendrimer, or equivalent structure).Contacting the polymer-nucleotide conjugate with a polymerase and aprimed target nucleic acid may result in the formation of a ternarycomplex which may be detected and in turn achieve a more accuratedetermination of the bases of the target nucleic acid.

When the polymer-nucleotide conjugate is used in replacement of singleunconjugated or untethered nucleotide to form a complex with thepolymerase and the target nucleic acid, the local concentration of thenucleotide is increased many fold, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. Thepolymer-nucleotide conjugate described herein can include at least onepolymer-nucleotide conjugate for interacting with the target nucleicacid. The multivalent composition can also include two, three, or fourdifferent polymer-nucleotide conjugates, each having a differentnucleotide conjugated to the particle.

In a polymer-nucleotide conjugate having a polymer-nucleotide conjugateform or a core-nucleotide conjugate form, multiple copies of the samenucleotide may be covalently bound to or noncovalently bound to theparticle. Examples of the particle can include a branched polymer; adendrimer; a cross linked polymer particle such as an agarose,polyacrylamide, acrylate, methacrylate, cyanoacrylate, methylmethacrylate particle; a glass particle; a ceramic particle; a metalparticle; a quantum dot; a liposome; an emulsion particle, or any otherparticle (e.g., nanoparticles, microparticles, or the like) known in theart. In a preferred embodiment, the particle is a branched polymer.

The nucleotide can be linked to the particle or core through a linker,and the nucleotide can be attached to one end or location of a polymer.The nucleotide can be conjugated to the particle through the base or the5′ end of the nucleotide. In some polymer-nucleotide conjugates, onenucleotide attached to one end or location of a polymer. In somepolymer-nucleotide conjugate, multiple nucleotides are attached to oneend or location of a polymer. The conjugated nucleotide is stericallyaccessible to one or more proteins, one or more enzymes, and nucleotidebinding moieties. In some embodiments, a nucleotide may be providedseparately from a nucleotide binding moiety such as a polymerase. Insome embodiments, the linker does not comprise a photo emitting or photoabsorbing group.

The particle or core can also have a binding moiety. In someembodiments, particles or cores may self-associate without the use of aseparate interaction moiety. In some embodiments, particles or cores mayself-associate due to buffer conditions or salt conditions, e.g., as inthe case of calcium-mediated interactions of hydroxyapatite particles,lipid or polymer mediated interactions of micelles or liposomes, orsalt-mediated aggregation of metallic (such as iron or gold)nanoparticles.

The polymer-nucleotide conjugates can have one or more labels (e.g.,detectable reporter moieties). Examples of the labels include but arenot limited to fluorophores, spin labels, metals or metal ions,colorimetric labels, nanoparticles, PET labels, radioactive labels, orother such label as may render said composition detectable by suchmethods as are known in the art of the detection of macromolecules ormolecular interactions. The label may be attached to the nucleotide(e.g. by attachment to the base or the 5′ phosphate moiety of anucleotide), to the particle itself (e.g., to the PEG subunits) or tothe core (e.g., to the streptavidin core), to an end of the polymer, toa central moiety, or to any other location within saidpolymer-nucleotide conjugate which would be recognized by one of skillin the art to be sufficient to render said composition, such as aparticle, detectable by such methods as are known in the art ordescribed elsewhere herein. In some embodiments, one or more labels areprovided so as to correspond to or differentiate a particularpolymer-nucleotide conjugate.

One example of the polymer-nucleotide conjugate (e.g.,polymer-nucleotide conjugate) is a polymer-nucleotide conjugate.Examples of the branched polymer include polyethylene glycol (PEG),polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolicacid, polyglycine, polyvinyl acetate, a dextran, or other such polymers.In one embodiment, the polymer is a PEG. In another embodiment, thepolymer can have PEG branches.

Suitable polymers may be characterized by a repeating unit having afunctional group suitable for derivatization such as an amine, ahydroxyl, a carbonyl, or an allyl group. The polymer can also have oneor more pre-derivatized substituents such that one or more particularsubunits comprise a site of derivatization or a branch site, whether ornot other subunits include the same site, substituent, or moiety. Apre-derivatized substituent may comprise or may further comprise, forexample, a nucleotide, a nucleoside, a nucleotide analog, a label suchas a fluorescent label, radioactive label, or spin label, an interactionmoiety, an additional polymer moiety, or the like, or any combination ofthe foregoing.

In the polymer-nucleotide conjugate (e.g., polymer-nucleotideconjugate), the polymer can have a plurality of branches. The branchedpolymer can have various configurations, including but are not limitedto stellate (“starburst”) forms, aggregated stellate (“helter skelter”)forms, bottle brush, or dendrimer. The branched polymer can radiate froma central attachment point or central moiety, or may include multiplebranch points, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morebranch points. In some embodiments, each subunit of a polymer mayoptionally constitute a separate branch point.

In the polymer-nucleotide conjugate, the length and size of the branchcan differ based on the type of polymer. In some branched polymers, thebranch may have a length of between 1 and 1,000 nm, between 1 and 100nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm,between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm,between 1 and 800 nm, or between 1 and 900 nm, or more, or having alength falling within or between any of the values disclosed herein. Insome branched polymers, the branch may have a size corresponding to anapparent molecular weight of 1K, 2K, 3K, 4K, 5 K, 10 K, 15 K, 20 K, 30K, 50 K, 80 K, 100 K, or any value within a range defined by any two ofthe foregoing. The apparent molecular weight of a polymer may becalculated from the known molecular weight of a representative number ofsubunits, as determined by size exclusion chromatography, as determinedby mass spectrometry, or as determined by any other method as is knownin the art. The polymer can have multiple branches. The number ofbranches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64,128 or more, or a number falling within a range defined by any two ofthese values.

For the polymer-nucleotide conjugate, the branched polymer of 4, 8, 16,32, or 64 branches can have nucleotides attached to the ends of PEGbranches, such that each end has attached thereto 0, 1, 2, 3, 4, 5, 6 ormore nucleotides. In one non-limiting example, the branched polymer ofbetween 3 and 128 PEG arms having attached to the polymer branches endsone or more nucleotides, such that each end has attached thereto 0, 1,2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs. In someembodiments, a branched polymer or dendrimer has an even number of arms.In some embodiments, a branched polymer or dendrimer has an odd numberof arms.

In the polymer-nucleotide conjugate, each branch or a subset of branchesof the polymer may have attached thereto a moiety comprising anucleotide (e.g., an adenine, a thymine, a uracil, a cytosine, or aguanine residue or a derivative or mimetic thereof), and the moiety iscapable of binding to a polymerase, reverse transcriptase, or othernucleotide binding domain. Optionally, the nucleotide moiety may becapable of binding to a polymerase-template-primer complex but notincorporate, or can incorporate into an elongating nucleic acid chainduring a polymerase reaction. In some embodiments, the nucleotide moietycomprises a chain terminating moiety which blocks incorporation of asubsequent nucleotide during a polymerase-mediated reaction. In someembodiments, the nucleotide moiety may be unblocked (reversibly blocked)such that a subsequent nucleotide is not capable of being incorporatedinto an elongating nucleic acid chain during a polymerase reaction untilsuch block is removed, after which the subsequent nucleotide is thencapable of being incorporated into an elongating nucleic acid chainduring a polymerase reaction.

The polymer-nucleotide conjugate can further have a binding moiety ineach branch or a subset of branches. Some examples of the binding moietyinclude but are not limited to biotin, avidin, streptavidin or the like,polyhistidine domains, complementary paired nucleic acid domains,G-quartet forming nucleic acid domains, calmodulin, maltose-bindingprotein, cellulase, maltose, sucrose, glutathione-S-transferase,glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine andderivatives thereof, benzylcysteine and derivatives thereof, anantibody, an epitope, a protein A, a protein G. The binding moiety canbe any interactive molecules or fragment thereof known in the art tobind to or facilitate interactions between proteins, between proteinsand ligands, between proteins and nucleic acids, between nucleic acids,or between small molecule interaction domains or moieties.

In some embodiments, the polymer-nucleotide conjugate may comprise oneor more elements of a complementary interaction moiety. Exemplarycomplementary interaction moieties include, for example, biotin andavidin; SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC andProtein A, Protein G, ProteinA/G, or Protein L; maltose binding proteinand maltose; lectin and cognate polysaccharide; ion chelation moieties,complementary nucleic acids, nucleic acids capable of forming triplex ortriple helical interactions; nucleic acids capable of formingG-quartets, and the like. One of skill in the art will readily recognizethat many pairs of moieties exist and are commonly used for theirproperty of interacting strongly and specifically with one another; andthus any such complementary pair or set is considered to be suitable forthis purpose in constructing or envisioning the compositions of thepresent disclosure. In some embodiments, a composition as disclosedherein may comprise compositions in which one element of a complementaryinteraction moiety is attached to one molecule or multivalent ligand,and the other element of the complementary interaction moiety isattached to a separate molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to a single molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to separate arms of, or locations on, a single molecule ormultivalent ligand. In some embodiments, a composition as disclosedherein may comprise compositions in which both or all elements of acomplementary interaction moiety are attached to the same arm of, orlocations on, a single molecule or multivalent ligand. In someembodiments, compositions comprising one element of a complementaryinteraction moiety and compositions comprising another element of acomplementary interaction moiety may be simultaneously or sequentiallymixed. In some embodiments, interactions between molecules or particlesas disclosed herein allow for the association or aggregation of multiplemolecules or particles such that, for example, detectable signals areincreased. In some embodiments, fluorescent, colorimetric, orradioactive signals are enhanced. In other embodiments, otherinteraction moieties as disclosed herein or as are known in the art arecontemplated. In some embodiments, a composition as provided herein maybe provided such that one or more molecules comprising a firstinteraction moiety such as, for example, one or more imidazole orpyridine moieties, and one or more additional molecules comprising asecond interaction moiety such as, for example, histidine residues, aresimultaneously or sequentially mixed. In some embodiments, saidcomposition comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridinemoieties. In some embodiments, said composition comprises 1, 2, 3, 4, 5,6, or more histidine residues. In such embodiments, interaction betweenthe molecules or particles as provided may be facilitated by thepresence of a divalent cation such as nickel, manganese, magnesium,calcium, strontium, or the like. In some embodiments, for example, a(His)3 group may interact with a (His)3 group on another molecule orparticle via coordination of a nickel or manganese ion.

The polymer-nucleotide conjugate may comprise one or more buffers,salts, ions, or additives. In some embodiments, representative additivesmay include, but are not limited to, betaine, spermidine, detergentssuch as Triton X-100, Tween 20, SDS, or NP-40, ethylene glycol,polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol,methylcellulose, heparin, heparan sulfate, glycerol, sucrose,1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol,propylene glycol, polypropylene glycol, block copolymers such as thePluronic (r) series polymers, arginine, histidine, imidazole, or anycombination thereof, or any substance known in the art as a DNA“relaxer” (a compound, with the effect of altering the persistencelength of DNA, altering the number of within-polymer junctions orcrossings, or altering the conformational dynamics of a DNA moleculesuch that the accessibility of sites within the strand to DNA bindingmoieties is increased).

The polymer-nucleotide conjugate may include zwitterionic compounds asadditives. Further representative additives may be found in Lorenz, T.C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which is herebyincorporated by reference with respect to its disclosure of additivesfor the facilitation of nucleic acid binding or dynamics, or thefacilitation of processes involving the manipulation, use, or storage ofnucleic acids.

In some embodiments, the multivalent binding compositions include atleast one cations may include, but are not limited to, sodium,magnesium, strontium, barium, potassium, manganese, calcium, lithium,nickel, cobalt, or other such cations as are known in the art tofacilitate nucleic acid interactions, such as self-association,secondary or tertiary structure formation, base pairing, surfaceassociation, peptide association, protein binding, or the like.

When the polymer-nucleotide conjugate is used to replace an unconjugatedor untethered nucleotide to form a complex with the polymerase and thetarget nucleic acid, the local concentration of the nucleotide isincreased many folds, which in turn enhances the signal intensity,particularly the correct signal versus mismatch. The present disclosurecontemplates contacting the polymer-nucleotide conjugate with apolymerase and a primed target nucleic acid to determine the formationof a ternary binding complex.

Because of the increased local concentration of the nucleotide on thepolymer-nucleotide conjugate, the binding between the polymerase, theprimed target strand, and the nucleotide, when the nucleotide iscomplementary to the next base of the target nucleic acid, becomes morefavorable. The formed binding complex has a longer persistence timewhich in turn helps shorten the imaging step. The high signal intensityresulted from the use of the polymer-nucleotide conjugate remain for theentire binding and imaging step. The strong binding between thepolymerase, the primed target strand, and the nucleotide or nucleotideanalog also means that the formed binding complex will remain stabilizedduring the washing step and the signal will remain at a high intensitywhen other reaction mixture and unmatched nucleotide analogs are washedaway. After the imaging step, the binding complex can be destabilizedand the primed target nucleic acid can then be extended for one base.After the extension, the binding and imaging steps can be repeated againwith the use of the polymer-nucleotide conjugate to determine theidentity of the next base.

The compositions and methods of the present disclosure provide a robustand controllable means of establishing and maintaining a ternary enzymecomplex (e.g., during sequencing), as well as providing vastly improvedmeans by which the presence of said complex may be identified and/ormeasured, and a means by which the persistence of said complex may becontrolled. This provides important solutions to problems such as thatof determining the identity of the N+1 base in nucleic acid sequencingapplications.

Without intending to be bound by any particular theory, it has beenobserved that multivalent binding compositions disclosed hereinassociate with polymerase nucleotide complexes in order to form aternary binding complexes with a rate that is time-dependent, thoughsubstantially slower than the rate of association known to be obtainableby nucleotides in free solution. Thus, the on-rate (Kon) issubstantially and surprisingly slower than the on rate for singlenucleotides or nucleotides not attached to multivalent ligand complexes.Importantly, however, the off rate (Koff) of the multivalent ligandcomplex is substantially slower than that observed for nucleotides infree solution. Therefore, the multivalent ligand complexes of thepresent disclosure provide a surprising and beneficial improvement ofthe persistence of ternary polymerase-polynucleotide-nucleotidecomplexes (especially over such complexes that are formed with freenucleotides) allowing, for example, significant improvements in imagingquality for nucleic acid sequencing applications, over currentlyavailable methods and reagents. Importantly, this property of themultivalent substrates disclosed herein renders the formation of visibleternary complexes controllable, such that subsequent visualization,modification, or processing steps may be undertaken essentially withoutregard to the dissociation of the complex—that is, the complex can beformed, imaged, modified, or used in other ways as necessary, and willremain stable until a user carries out an affirmative dissociation step,such as exposing the complexes to a dissociation buffer.

In various embodiments, polymerases suitable for the binding interaction(e.g., during sequencing) describe herein include may include anypolymerase as is or may be known in the art. Exemplary polymerases mayinclude but are not limited to: Klenow DNA polymerase, Thermus aquaticusDNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophageT7 DNA polymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase IIIalpha and epsilon; 9 degree N polymerase, reverse transcriptases such asHIV type M or O reverse transcriptases, avian myeloblastosis virusreverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, or telomerase. Further non-limiting examples of DNApolymerases can include those from various Archaea genera, such as,Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus,Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus,Thermococcus, and Vulcanisaeta and the like or variants thereof,including such polymerases as are known in the art such as Vent™, DeepVent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases. In someembodiments, the polymerase is a Klenow polymerase.

The ternary complex has longer persistence time when the nucleotide onthe polymer-nucleotide conjugate is complementary to the target nucleicacid than when non-complementary. The ternary complex also has longerpersistence time when the nucleotide on the polymer-nucleotide conjugateis complementary to the target nucleic acid than a complementarynucleotide that is not conjugated or tethered. For example, in someembodiments, said ternary complexes may have a persistence time of lessthan 1 s, greater than 1 s, greater than 2 s, greater than 3 s, greaterthan 5 s, greater than 10 s, greater than 15 s, greater than 20 s,greater than 30 s, greater than 60 s, greater than 120 s, greater than360 s, greater than 3600 s, or more, or for a time lying within a rangedefined by any two or more of these values.

The persistence time can be measured, for example, by observing theonset and/or duration of a binding complex, such as by observing asignal from a labeled component of the binding complex. For example, alabeled nucleotide or a labeled reagent comprising one or morenucleotides may be present in a binding complex, thus allowing thesignal from the label to be detected during the persistence time of thebinding complex.

It has been observed that different ranges of persistence times areachievable with different salts or ions, showing, for example, thatcomplexes formed in the presence of, for example, magnesium form morequickly than complexes formed with other ions. It has also been observedthat complexes formed in the presence of, for example, strontium, formreadily and dissociate completely or with substantial completeness uponwithdrawal of the ion or upon washing with buffer lacking one or morecomponents of the present compositions, such as, e.g., a polymer and/orone or more nucleotides, and/or one or more interaction moieties, or abuffer containing, for example, a chelating agent which may cause oraccelerate the removal of a divalent cation from the multivalent reagentcontaining complex. Thus, in some embodiments, a composition of thepresent disclosure comprises magnesium. In some embodiments, acomposition of the present disclosure comprises calcium. In someembodiments, a composition of the present disclosure comprises strontiumor barium. In some embodiments, a composition of the present disclosurecomprises cobalt. In some embodiments, a composition of the presentdisclosure comprises MgCl₂. In some embodiments, a composition of thepresent disclosure comprises CaCl₂. In some embodiments, a compositionof the present disclosure comprises SrCl₂. In some embodiments, acomposition of the present disclosure comprises CoCl₂. In someembodiments, the composition comprises no, or substantially nomagnesium. In some embodiments, the composition comprises no, orsubstantially no calcium. In some embodiments, the methods of thepresent disclosure provide for the contacting of one or more nucleicacids with one or more of the compositions disclosed herein wherein saidcomposition lacks either one of calcium or magnesium, or lacks bothcalcium and magnesium.

The dissociation of ternary complexes can be controlled by changing thebuffer conditions. After the imaging step, a buffer with increased saltcontent is used to cause dissociation of the ternary complexes such thatlabeled polymer-nucleotide conjugates can be washed out, providing ameans by which signals can be attenuated or terminated, such as in thetransition between one sequencing cycle and the next. This dissociationmay be effected, in some embodiments, by washing the complexes with abuffer lacking a necessary metal or cofactor. In some embodiments, awash buffer may comprise one or more compositions for the purpose ofmaintaining pH control. In some embodiments, a wash buffer may compriseone or more monovalent cations, such as sodium. In some embodiments, awash buffer lacks or substantially lacks a divalent cation, for example,having no or substantially no strontium, calcium, magnesium, ormanganese. In some embodiments, a wash buffer further comprises achelating agent, such as, for example, EDTA, EGTA, nitrilotriaceticacid, polyhistidine, imidazole, or the like. In some embodiments, a washbuffer may maintain the pH of the environment at the same level as forthe bound complex. In some embodiments, a wash buffer may raise or lowerthe pH of the environment relative to the level seen for the boundcomplex. In some embodiments, the pH may be within a range from 2-4,2-7, 5-8, 7-9, 7-10, or lower than 2, or higher than 10, or a rangedefined by any two of the values provided herein.

Addition of a particular ion may affect the binding of the polymerase toa primed target nucleic acid, the formation of a ternary complex, thedissociation of a ternary complex, or the incorporation of one or morenucleotides into an elongating nucleic acid such as during a polymerasereaction. In some embodiments, relevant anions may comprise chloride,acetate, gluconate, sulfate, phosphate, or the like. In someembodiments, an ion may be included in the compositions of the presentdisclosure by the addition of one or more acids, bases, or salts, suchas NiCl₂, CoCl₂, MgCl₂, MnCl₂, SrCl₂, CaCl₂, CaSO₄, SrCO₃, BaCl₂ or thelike. Representative salts, ions, solutions and conditions may be foundin Remington: The Science and Practice of Pharmacy, 20th. Edition,Gennaro, A. R., Ed. (2000), which is hereby incorporated by reference inits entirety, and especially with respect to Chapter 17 and relateddisclosure of salts, ions, salt solutions, and ionic solutions.

The present disclosure contemplates contacting the polymer-nucleotideconjugate with one or more polymerases. The contacting can be optionallydone in the presence of one or more target nucleic acids. In someembodiments, said target nucleic acids are single stranded nucleicacids. In some embodiments, the target nucleic acids are hybridized to anucleic acid primer. In some embodiments, said target nucleic acids aredouble stranded nucleic acids. In some embodiments, said contactingcomprises contacting the polymer-nucleotide conjugate with onepolymerase. In some embodiments, said contacting comprises thecontacting of said composition comprising one or more nucleotides withmultiple polymerases. The polymerase can be bound to a single nucleicacid molecule.

The binding between target nucleic acid and polymer-nucleotide conjugatemay be provided in the presence of a polymerase that has been renderedcatalytically inactive. In one embodiment, the polymerase may have beenrendered catalytically inactive by mutation. In one embodiment, thepolymerase may have been rendered catalytically inactive by chemicalmodification. In some embodiments, the polymerase may have been renderedcatalytically inactive by the absence of a necessary substrate, ion, orcofactor. In some embodiments, the polymerase enzyme may have beenrendered catalytically inactive by the absence of magnesium ions.

The binding between target nucleic acid and polymer-nucleotide conjugateoccur in the presence of a polymerase wherein the binding solution,reaction solution, or buffer lacks a catalytic ion such as magnesium ormanganese. Alternatively, the binding between target nucleic acid andpolymer-nucleotide conjugate occur in the presence of a polymerasewherein the binding solution, reaction solution, or buffer comprises anon-catalytic ion such strontium, barium or calcium.

When the catalytically inactive polymerases are used to help a nucleicacid interact with a multivalent binding composition, the interactionbetween said composition and said polymerase stabilizes a ternarycomplex so as to render the complex detectable by fluorescence or byother methods as disclosed herein or otherwise known in the art. Unboundpolymer-nucleotide conjugates may optionally be washed away prior todetection of the ternary binding complex.

Contacting of one or more nucleic acids with the polymer-nucleotideconjugates disclosed herein in a solution containing either one ofcalcium or magnesium, or containing both calcium and magnesium.Alternatively, the contacting of one or more nucleic acids with thepolymer-nucleotide conjugates disclosed herein in a solution lackingeither one of calcium or magnesium, or lacking both calcium ormagnesium, and in a separate step, without regard to the order of thesteps, adding to the solution one of calcium or magnesium, or bothcalcium and magnesium. In some embodiments, the contacting of one ormore nucleic acids with the polymer-nucleotide conjugates disclosedherein in a solution lacking strontium or barium, and comprises in aseparate step, without regard to the order of the steps, adding to thesolution strontium.

Disclosed herein are polymer-nucleotide conjugates and their use inanalyzing nucleic acid including sequencing or other bioassayapplications. An increase in binding of a nucleotide to an enzyme (e.g.,polymerase) or an enzyme complex can be effected by increasing theeffective concentration of the nucleotide. The increase can be achievedby increasing the concentration of the nucleotide in free solution, orby increasing the amount of the nucleotide in proximity to the relevantbinding site. The increase can also be achieved by physicallyrestricting a number of nucleotides into a limited volume thus resultingin a local increase in concentration, and such as structure may thusbind to the binding site with a higher apparent avidity than would beobserved with unconjugated, untethered, or otherwise unrestrictedindividual nucleotide. One exemplary means of effecting such restrictionis by providing a polymer-nucleotide conjugate in which multiplenucleotides are bound to a particle such as a polymer, a branchedpolymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

The polymer-nucleotide conjugate disclosed herein can include aplurality of nucleotide moieties attached to the particle. In someembodiments, the plurality of nucleotides moieties is comprised of thesame type of nucleotide moiety (e.g., having the same or similar basepairing properties). When the plurality of nucleotide moieties iscomplementary to the next nucleotide in a target nucleic acid to beidentified, the polymer-nucleotide conjugate forms a binding complex(multivalent binding complex) between at least two nucleotide moietiesand next nucleotide in at least two copies of the target nucleic acidsequence. In some embodiments, the multivalent binding complex comprisestwo or more polymerases that associate with the primed template of thetarget nucleic acid molecule. The multivalent binding complexesdescribed herein exhibits increased stability and longer persistencetime than the binding complex formed using a single unconjugated oruntethered nucleotide. When bound to a polymerase, the multivalentbinding complex can withstanding washing steps, so that the signalintensity remains high throughout the imaging and washing steps of theworkflow, see for e.g., in FIG. 7. The polymer core of thepolymer-nucleotide conjugate can be labeled with two or more detectablelabels, which at least partially contributes to the enhanced signal thatcan be detected.

In some embodiments, the at least one polymer-nucleotide conjugatecomprises two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, as shown for example, in FIG. 5A andFIG. 5B. In some embodiments, the polymer-nucleotide conjugatecomprises: (a) a core, and (b) a plurality of nucleotide arms where eachnucleotide arm comprises (i) a core attachment moiety, (ii) a spacercomprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, asshown for example in FIG. 5A-D and FIG. 6A-C.

In some embodiments, the spacer is attached to the linker, wherein thelinker is attached to the nucleotide unit. In some embodiments, thenucleotide unit comprises a base, sugar and at least one phosphategroup. In some embodiments, the linker is attached to the nucleotideunit through the base. In some embodiments, the linker comprises analiphatic chain or an oligo ethylene glycol chain where both linkerchains having 2-6 subunits and optionally the linker includes anaromatic moiety (FIG. 6A, FIG. 6B, and FIG. 6C). In some embodiments,the polymer-nucleotide conjugate comprises a core attached to multiplenucleotide arms, and wherein the multiple nucleotide arms have the sametype of nucleotide unit which is selected from a group consisting ofdATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the low-bindingsupport further comprises a plurality of polymer-nucleotide conjugateswhich includes a mixture of polymer-nucleotide conjugates having two ormore different types of nucleotides selected from a group consisting ofdATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the polymer-nucleotide conjugate comprises a coreattached to multiple nucleotide arms, wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position. In some embodiments, the chainterminating moiety is selected from a group consisting of an alkylgroup, alkenyl group, alkynyl group, allyl group, aryl group, benzylgroup, azide group, amine group, amide group, keto group, isocyanategroup, phosphate group, thio group, disulfide group, carbonate group,urea group, or silyl group.

In some embodiments, the chain terminating moiety comprises a 3′-O-alkylhydroxylamino group, a 3′-phosphorothioate group, a 3′-O-malonyl group,or a 3′-O-benzyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof. In someembodiments, the chain-terminating moiety comprises an azide, azido orazidomethyl group.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide arm, for example with a chemical compound, light orheat. In some embodiments, the chain terminating moiety comprises analkyl, alkenyl, alkynyl or allyl group which are cleavable withtetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄), with piperidine,or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In someembodiment, the chain terminating moiety comprises an aryl or benzylgroup which are cleavable with Pd/C. In some embodiments, the chainterminating moiety comprises an amine, amide, keto, isocyanate,phosphate, thio or disulfide group which are cleavable with phosphine orwith a thiol group including beta-mercaptoethanol or dithiothritol(DTT). In some embodiments, the chain terminating moiety comprises acarbonate group which is cleavable with potassium carbonate (K₂CO₃) inMeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH).In some embodiments, the chain terminating moiety comprises a urea orsilyl group which are cleavable with tetrabutylammonium fluoride,pyridine-HF, with ammonium fluoride, or with triethylaminetrihydrofluoride. In some embodiments, the chain terminating moiety isan azide, azido or azidomethyl group which are cleavable with aphosphine compound. In some embodiments, the phosphine compoundcomprises a derivatized tri-alkyl phosphine moiety or a derivatizedtri-aryl phosphine moiety. In some embodiments, the phosphine compoundcomprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenylphosphine (BS-TPP).

In some embodiments, the polymer-nucleotide conjugate comprises a coreattached to multiple nucleotide arms, wherein the core or the nucleotidebase comprises a label. In some embodiments, the label is a detectablereporter moiety. The polymer-nucleotide conjugate can have one or morelabels. Examples of the detectable reporter moiety include but are notlimited to fluorophores, spin labels, metals or metal ions, colorimetriclabels, nanoparticles, PET labels, radioactive labels, or other suchlabel as may render said composition detectable by such methods as areknown in the art of the detection of macromolecules or molecularinteractions. The detectable reporter moiety may be attached to thenucleotide (e.g. by attachment to the 5′ phosphate moiety of anucleotide), to the particle itself (e.g., to the PEG subunits), to anend of the polymer, to a central moiety, or to any other location withinsaid polymer-nucleotide conjugate which would be recognized by one ofskill in the art to be sufficient to render said composition, such as aparticle, detectable by such methods as are known in the art ordescribed elsewhere herein. In some embodiments, one or more labels areprovided so as to correspond to or differentiate a particularpolymer-nucleotide conjugate. The detectable reporter moiety can be afluorophore. In some embodiments, the core can be an avidin-like moietyand the core attachment moiety can be a biotin moiety.

Exemplary polymer-nucleotide conjugates and methods of use are describedin U.S. application Ser. No. 16/579,794, filed Sep. 23, 2019, thecontents of the aforementioned patent application is hereby expresslyincorporated by reference for all purposes.

The polymer-nucleotide conjugate (polymer-nucleotide conjugate) can beused to localize detectable signals to active regions of biochemicalinteractions, such as sites of protein-nucleic acid interactions,nucleic acid hybridization reactions, or enzymatic reactions, such aspolymerase reactions. For example, the polymer-nucleotide conjugatesdescribed herein can be utilized to identify sites of base binding to atemplate or base incorporation in elongating nucleic acid chains duringpolymerase reactions and to provide base discrimination for sequencingand array based applications. The increased binding between the targetnucleic acid and the nucleotide in the multivalent binding composition,when the nucleotide is complementary to the target nucleic acid,provides enhanced signal that greatly improve base call accuracy andshorten imaging time.

In addition, the use of polymer-nucleotide conjugates allows sequencingsignals from a given sequence to originate within cluster regionscontaining multiple copies of the target sequence. Sequencing methodsthat include multiple copies of a target sequence (e.g., concatemer)have the advantage that signals can be amplified due to the presence ofmultiple simultaneous sequencing reactions within the defined region,each providing its own signal. The presence of multiple signals within adefined area also reduces the impact of any single skipped cycle, due tothe fact that the signal from a large number of correct base calls canoverwhelm the signal from a smaller number of skipped or incorrect basecalls, therefore providing methods for reducing phasing errors and/or toimprove read length in sequencing reactions.

The polymer-nucleotide conjugates and their use disclosed herein lead toone or more of: (i) stronger signal for better base-calling accuracycompared to conventional nucleic acid amplification and sequencingmethodologies; (ii) allow greater discrimination of sequence-specificsignal from background signals; (iii) reduced requirements for theamount of starting material necessary, (iv) increased sequencing rateand shortened sequencing time; (v) reducing phasing errors, and (vi)improving read length in sequencing reactions.

One of ordinary skill would recognize that in a series of iterativesequencing reactions, occasionally one or more sites will fail toincorporate a nucleotide during a given cycle, thus leading one or moresites to be unsynchronized with the bulk of the elongating nucleic acidchains. Under conditions in which sequencing signals are derived fromreactions occurring on single copies of a target nucleic acid, thesefailures to incorporate will yield discrete errors in the outputsequence. Use of the polymer-nucleotide conjugates for sequencing canreduce this type of error in sequencing reactions. For example, the useof multivalent substrates that are capable of binding to apolymerase-template-primer complex, or capable of incorporation into theelongating strand, by providing increased probabilities of rebindingupon premature dissociation of a ternary polymerase complex, can reducethe frequency of “skipped” cycles in which a base is not incorporated.Thus, in some embodiments, the present disclosure contemplates the useof multivalent substrates as disclosed herein comprising a nucleotidehaving a free, or reversibly modified, 5′ phosphate, diphosphate, ortriphosphate moiety, and wherein the nucleotide is connected to theparticle or polymer as disclosed herein, through a labile or cleavablelinkage. In some embodiments, the present disclosure contemplates areduction in the intrinsic error rate due to skipped incorporations as aresult of the use of the multivalent substrates disclosed herein.

The present disclosure also contemplates sequencing reactions in whichsequencing signals from or relating to a given sequence are derived fromor originate within definable regions containing multiple copies of thetarget sequence. Sequencing methods incorporating multiple copies of atarget sequence have the advantage that signals can be amplified due tothe presence of multiple simultaneous sequencing reactions within thedefined region, each providing its own signal. The presence of multiplesignals within a defined area also reduces the impact of any singleskipped cycle, due to the fact that the signal from a large number ofcorrect base calls can overwhelm the signal from a smaller number ofskipped or incorrect base calls. The present disclosure furthercontemplates the inclusion of free, unlabeled nucleotides duringelongation reactions, or during a separate part of the elongation cycle,in order to provide incorporation at sites that may have been skipped inprevious cycles. For example, during or following an incorporationcycle, unlabeled blocked nucleotides may be added such that they may beincorporated at skipped sites. The unlabeled blocked nucleotides may beof the same type or types as the nucleotide attached to the multivalentbinding substrate or substrates that are or were present during aparticular cycle, or a mixture of 1, 2, 3, 4 or more types of unlabeledblocked nucleotides may be included.

When each sequencing cycle proceeds perfectly, each reaction within thedefined region will provide an identical signal. However, as notedelsewhere herein, in a series of iterative sequencing reactions,occasionally one or more sites will fail to incorporate a nucleotideduring a given cycle, thus leading one or more sites to beunsynchronized with the bulk of the elongating nucleic acid chains. Thisissue, referred to as “phasing,” leads to degradation of the sequencingsignal as the signal is contaminated with spurious signals from siteshaving skipped one or more cycles. This, in turn, creates the potentialfor errors in base identification. The progressive accumulation ofskipped cycles through multiple cycles also reduces the effective readlength, due to progressive degradation of the sequencing signal witheach cycle. It is a further object of this disclosure to provide methodsfor reducing phasing errors and/or to improve read length in sequencingreactions.

The sequencing method can include contacting a target nucleic acid ormultiple target nucleic acids, comprising multiple linked or unlinkedcopies of a target sequence, with the multivalent binding compositionsdescribed herein. Contacting said target nucleic acid, or multipletarget nucleic acids comprising multiple linked or unlinked copies of atarget sequence, with one or more polymer-nucleotide conjugates mayprovide a substantially increased local concentration of the correctnucleotide being interrogated in a given sequencing cycle, thussuppressing signals from improper incorporations or phased nucleic acidchains (i.e., those elongating nucleic acid chains which have had one ormore skipped cycles).

Methods of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said target nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more polymer-nucleotide conjugates. This method results in areduction in the error rate of sequencing as indicated by reduction inthe misidentification of bases, the reporting of nonexistent bases, orthe failure to report correct bases. In some embodiments, said reductionin the error orate of sequencing may comprise a reduction of 5%, 10%,15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the errorrate observed using monovalent ligands, including free nucleotides,labeled free nucleotides, protein or peptide bound nucleotides, orlabeled protein or peptide bound nucleotides.

The method of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said templet nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more polymer-nucleotide conjugates. This method results in anincrease in average read length of 5%, 10%, 15%, 20% 25%, 50%, 75%,100%, 150%, 200%, 300%, or more compared to the average read lengthobserved using monovalent ligands, including free nucleotides, labeledfree nucleotides, protein or peptide bound nucleotides, or labeledprotein or peptide bound nucleotides.

Methods of obtaining nucleic acid sequence information, said methodscomprising contacting a target nucleic acid, or multiple target nucleicacids, wherein said target nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more polymer-nucleotide conjugates. This method results in anincrease in average read length of 10, 20, 25, 30, 50, 75, 100, 125,150, 200, 250, 300, 350, 400, 500 nucleotides, or more compared to theaverage read length observed using monovalent ligands, including freenucleotides, labeled free nucleotides, protein or peptide boundnucleotides, or labeled protein or peptide bound nucleotides.

The use of the polymer-nucleotide conjugates for sequencing effectivelyshortens the sequencing time. The sequencing reaction cycle comprisingthe contacting, detecting, and incorporating steps is performed in atotal time ranging from about 5 minutes to about 60 minutes. In someembodiments, the sequencing reaction cycle is performed in at least 5minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes,at least 40 minutes, at least 50 minutes, or at least 60 minutes. Insome embodiments, the sequencing reaction cycle is performed in at most60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes,at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome embodiments the sequencing reaction cycle may be performed in atotal time ranging from about 10 minutes to about 30 minutes. Those ofskill in the art will recognize that the sequencing cycle time may haveany value within this range, e.g., about 16 minutes.

The use of the polymer-nucleotide conjugates for sequencing provides anmore accuracy base readout. The disclosed compositions and methods fornucleic acid sequencing will provide an average Q-score for base-callingaccuracy over a sequencing run that ranges from about 20 to about 50. Insome embodiments, the average Q-score is at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50. Thoseof skill in the art will recognize that the average Q-score may have anyvalue within this range, e.g., about 32. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 30 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 35for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 40 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 45for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

The present disclosure relates to polymer-nucleotide conjugates eachhaving a plurality of nucleotides conjugated to a particle or core(e.g., a polymer, branched polymer, dendrimer, or equivalent structure).Contacting the polymer-nucleotide conjugate with a polymerase and aprimed target nucleic acid may result in the formation of a ternarycomplex which may be detected and in turn achieve a more accuratedetermination of the bases of the target nucleic acid.

When the polymer-nucleotide conjugate is used in replacement of singleunconjugated or untethered nucleotide to form a complex with thepolymerase and the target nucleic acid, the local concentration of thenucleotide is increased many fold, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. Thepolymer-nucleotide conjugate described herein can include at least onepolymer-nucleotide conjugate for interacting with the target nucleicacid. The multivalent composition can also include two, three, or fourdifferent polymer-nucleotide conjugates, each having a differentnucleotide conjugated to the particle.

In a polymer-nucleotide conjugate having a polymer-nucleotide conjugateform or a core-nucleotide conjugate form, multiple copies of the samenucleotide may be covalently bound to or noncovalently bound to theparticle. Examples of the particle can include a branched polymer; adendrimer; a cross linked polymer particle such as an agarose,polyacrylamide, acrylate, methacrylate, cyanoacrylate, methylmethacrylate particle; a glass particle; a ceramic particle; a metalparticle; a quantum dot; a liposome; an emulsion particle, or any otherparticle (e.g., nanoparticles, microparticles, or the like) known in theart. In a preferred embodiment, the particle is a branched polymer.

The nucleotide can be linked to the particle or core through a linker,and the nucleotide can be attached to one end or location of a polymer.The nucleotide can be conjugated to the particle through the base or the5′ end of the nucleotide. In some polymer-nucleotide conjugates, onenucleotide attached to one end or location of a polymer. In somepolymer-nucleotide conjugate, multiple nucleotides are attached to oneend or location of a polymer. The conjugated nucleotide is stericallyaccessible to one or more proteins, one or more enzymes, and nucleotidebinding moieties. In some embodiments, a nucleotide may be providedseparately from a nucleotide binding moiety such as a polymerase. Insome embodiments, the linker does not comprise a photo emitting or photoabsorbing group.

The particle or core can also have a binding moiety. In someembodiments, particles or cores may self-associate without the use of aseparate interaction moiety. In some embodiments, particles or cores mayself-associate due to buffer conditions or salt conditions, e.g., as inthe case of calcium-mediated interactions of hydroxyapatite particles,lipid or polymer mediated interactions of micelles or liposomes, orsalt-mediated aggregation of metallic (such as iron or gold)nanoparticles.

The polymer-nucleotide conjugates can have one or more labels (e.g.,detectable reporter moieties). Examples of the labels include but arenot limited to fluorophores, spin labels, metals or metal ions,colorimetric labels, nanoparticles, PET labels, radioactive labels, orother such label as may render said composition detectable by suchmethods as are known in the art of the detection of macromolecules ormolecular interactions. The label may be attached to the nucleotide(e.g. by attachment to the base or the 5′ phosphate moiety of anucleotide), to the particle itself (e.g., to the PEG subunits) or tothe core (e.g., to the streptavidin core), to an end of the polymer, toa central moiety, or to any other location within saidpolymer-nucleotide conjugate which would be recognized by one of skillin the art to be sufficient to render said composition, such as aparticle, detectable by such methods as are known in the art ordescribed elsewhere herein. In some embodiments, one or more labels areprovided so as to correspond to or differentiate a particularpolymer-nucleotide conjugate.

One example of the polymer-nucleotide conjugate (e.g.,polymer-nucleotide conjugate) is a polymer-nucleotide conjugate.Examples of the branched polymer include polyethylene glycol (PEG),polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolicacid, polyglycine, polyvinyl acetate, a dextran, or other such polymers.In one embodiment, the polymer is a PEG. In another embodiment, thepolymer can have PEG branches.

Suitable polymers may be characterized by a repeating unit having afunctional group suitable for derivatization such as an amine, ahydroxyl, a carbonyl, or an allyl group. The polymer can also have oneor more pre-derivatized substituents such that one or more particularsubunits comprise a site of derivatization or a branch site, whether ornot other subunits include the same site, substituent, or moiety. Apre-derivatized substituent may comprise or may further comprise, forexample, a nucleotide, a nucleoside, a nucleotide analog, a label suchas a fluorescent label, radioactive label, or spin label, an interactionmoiety, an additional polymer moiety, or the like, or any combination ofthe foregoing.

In the polymer-nucleotide conjugate (e.g., polymer-nucleotideconjugate), the polymer can have a plurality of branches. The branchedpolymer can have various configurations, including but are not limitedto stellate (“starburst”) forms, aggregated stellate (“helter skelter”)forms, bottle brush, or dendrimer. The branched polymer can radiate froma central attachment point or central moiety, or may include multiplebranch points, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morebranch points. In some embodiments, each subunit of a polymer mayoptionally constitute a separate branch point.

In the polymer-nucleotide conjugate, the length and size of the branchcan differ based on the type of polymer. In some branched polymers, thebranch may have a length of between 1 and 1,000 nm, between 1 and 100nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm,between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm,between 1 and 800 nm, or between 1 and 900 nm, or more, or having alength falling within or between any of the values disclosed herein. Insome branched polymers, the branch may have a size corresponding to anapparent molecular weight of 1 K, 2 K, 3 K, 4 K, 5 K, 10 K, 15 K, 20 K,30 K, 50 K, 80 K, 100 K, or any value within a range defined by any twoof the foregoing. The apparent molecular weight of a polymer may becalculated from the known molecular weight of a representative number ofsubunits, as determined by size exclusion chromatography, as determinedby mass spectrometry, or as determined by any other method as is knownin the art. The polymer can have multiple branches. The number ofbranches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64,128 or more, or a number falling within a range defined by any two ofthese values.

For the polymer-nucleotide conjugate (e.g., polymer-nucleotideconjugate), the branched polymer of 4, 8, 16, 32, or 64 branches canhave nucleotides attached to the ends of PEG branches, such that eachend has attached thereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In onenon-limiting example, the branched polymer of between 3 and 128 PEG armshaving attached to the polymer branches ends one or more nucleotides,such that each end has attached thereto 0, 1, 2, 3, 4, 5, 6 or morenucleotides or nucleotide analogs. In some embodiments, a branchedpolymer or dendrimer has an even number of arms. In some embodiments, abranched polymer or dendrimer has an odd number of arms.

In the polymer-nucleotide conjugate (e.g., polymer-nucleotideconjugate), each branch or a subset of branches of the polymer may haveattached thereto a moiety comprising a nucleotide (e.g., an adenine, athymine, a uracil, a cytosine, or a guanine residue or a derivative ormimetic thereof), and the moiety is capable of binding to a polymerase,reverse transcriptase, or other nucleotide binding domain. Optionally,the nucleotide moiety may be capable of binding to apolymerase-template-primer complex but not incorporate, or canincorporate into an elongating nucleic acid chain during a polymerasereaction. In some embodiments, the nucleotide moiety comprises a chainterminating moiety which blocks incorporation of a subsequent nucleotideduring a polymerase-mediated reaction. In some embodiments, thenucleotide moiety may be unblocked (reversibly blocked) such that asubsequent nucleotide is not capable of being incorporated into anelongating nucleic acid chain during a polymerase reaction until suchblock is removed, after which the subsequent nucleotide is then capableof being incorporated into an elongating nucleic acid chain during apolymerase reaction.

The polymer-nucleotide conjugate can further have a binding moiety ineach branch or a subset of branches. Some examples of the binding moietyinclude but are not limited to biotin, avidin, streptavidin or the like,polyhistidine domains, complementary paired nucleic acid domains,G-quartet forming nucleic acid domains, calmodulin, maltose-bindingprotein, cellulase, maltose, sucrose, glutathione-S-transferase,glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine andderivatives thereof, benzylcysteine and derivatives thereof, anantibody, an epitope, a protein A, a protein G. The binding moiety canbe any interactive molecules or fragment thereof known in the art tobind to or facilitate interactions between proteins, between proteinsand ligands, between proteins and nucleic acids, between nucleic acids,or between small molecule interaction domains or moieties.

In some embodiments, the polymer-nucleotide conjugate may comprise oneor more elements of a complementary interaction moiety. Exemplarycomplementary interaction moieties include, for example, biotin andavidin; SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC andProtein A, Protein G, ProteinA/G, or Protein L; maltose binding proteinand maltose; lectin and cognate polysaccharide; ion chelation moieties,complementary nucleic acids, nucleic acids capable of forming triplex ortriple helical interactions; nucleic acids capable of formingG-quartets, and the like. One of skill in the art will readily recognizethat many pairs of moieties exist and are commonly used for theirproperty of interacting strongly and specifically with one another; andthus any such complementary pair or set is considered to be suitable forthis purpose in constructing or envisioning the compositions of thepresent disclosure. In some embodiments, a composition as disclosedherein may comprise compositions in which one element of a complementaryinteraction moiety is attached to one molecule or multivalent ligand,and the other element of the complementary interaction moiety isattached to a separate molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to a single molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to separate arms of, or locations on, a single molecule ormultivalent ligand. In some embodiments, a composition as disclosedherein may comprise compositions in which both or all elements of acomplementary interaction moiety are attached to the same arm of, orlocations on, a single molecule or multivalent ligand. In someembodiments, compositions comprising one element of a complementaryinteraction moiety and compositions comprising another element of acomplementary interaction moiety may be simultaneously or sequentiallymixed. In some embodiments, interactions between molecules or particlesas disclosed herein allow for the association or aggregation of multiplemolecules or particles such that, for example, detectable signals areincreased. In some embodiments, fluorescent, colorimetric, orradioactive signals are enhanced. In other embodiments, otherinteraction moieties as disclosed herein or as are known in the art arecontemplated. In some embodiments, a composition as provided herein maybe provided such that one or more molecules comprising a firstinteraction moiety such as, for example, one or more imidazole orpyridine moieties, and one or more additional molecules comprising asecond interaction moiety such as, for example, histidine residues, aresimultaneously or sequentially mixed. In some embodiments, saidcomposition comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridinemoieties. In some embodiments, said composition comprises 1, 2, 3, 4, 5,6, or more histidine residues. In such embodiments, interaction betweenthe molecules or particles as provided may be facilitated by thepresence of a divalent cation such as nickel, manganese, magnesium,calcium, strontium, or the like. In some embodiments, for example, a(His)3 group may interact with a (His)3 group on another molecule orparticle via coordination of a nickel or manganese ion.

The polymer-nucleotide conjugate may comprise one or more buffers,salts, ions, or additives. In some embodiments, representative additivesmay include, but are not limited to, betaine, spermidine, detergentssuch as Triton X-100, Tween 20, SDS, or NP-40, ethylene glycol,polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol,methylcellulose, heparin, heparan sulfate, glycerol, sucrose,1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol,propylene glycol, polypropylene glycol, block copolymers such as thePluronic (r) series polymers, arginine, histidine, imidazole, or anycombination thereof, or any substance known in the art as a DNA“relaxer” (a compound, with the effect of altering the persistencelength of DNA, altering the number of within-polymer junctions orcrossings, or altering the conformational dynamics of a DNA moleculesuch that the accessibility of sites within the strand to DNA bindingmoieties is increased).

The polymer-nucleotide conjugate may include zwitterionic compounds asadditives. Further representative additives may be found in Lorenz, T.C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which is herebyincorporated by reference with respect to its disclosure of additivesfor the facilitation of nucleic acid binding or dynamics, or thefacilitation of processes involving the manipulation, use, or storage ofnucleic acids.

In some embodiments, the multivalent binding compositions include atleast one cations may include, but are not limited to, sodium,magnesium, strontium, barium, potassium, manganese, calcium, lithium,nickel, cobalt, or other such cations as are known in the art tofacilitate nucleic acid interactions, such as self-association,secondary or tertiary structure formation, base pairing, surfaceassociation, peptide association, protein binding, or the like.

When the polymer-nucleotide conjugate is used to replace an unconjugatedor untethered nucleotide to form a complex with the polymerase and thetarget nucleic acid, the local concentration of the nucleotide isincreased many folds, which in turn enhances the signal intensity,particularly the correct signal versus mismatch. The present disclosurecontemplates contacting the polymer-nucleotide conjugate with apolymerase and a primed target nucleic acid to determine the formationof a ternary binding complex.

Because of the increased local concentration of the nucleotide on thepolymer-nucleotide conjugate, the binding between the polymerase, theprimed target strand, and the nucleotide, when the nucleotide iscomplementary to the next base of the target nucleic acid, becomes morefavorable. The formed binding complex has a longer persistence timewhich in turn helps shorten the imaging step. The high signal intensityresulted from the use of the polymer-nucleotide conjugate remain for theentire binding and imaging step. The strong binding between thepolymerase, the primed target strand, and the nucleotide or nucleotideanalog also means that the formed binding complex will remain stabilizedduring the washing step and the signal will remain at a high intensitywhen other reaction mixture and unmatched nucleotide analogs are washedaway. After the imaging step, the binding complex can be destabilizedand the primed target nucleic acid can then be extended for one base.After the extension, the binding and imaging steps can be repeated againwith the use of the polymer-nucleotide conjugate to determine theidentity of the next base.

The compositions and methods of the present disclosure provide a robustand controllable means of establishing and maintaining a ternary enzymecomplex (e.g., during sequencing), as well as providing vastly improvedmeans by which the presence of said complex may be identified and/ormeasured, and a means by which the persistence of said complex may becontrolled. This provides important solutions to problems such as thatof determining the identity of the N+1 base in nucleic acid sequencingapplications.

Without intending to be bound by any particular theory, it has beenobserved that multivalent binding compositions disclosed hereinassociate with polymerase nucleotide complexes in order to form aternary binding complexes with a rate that is time-dependent, thoughsubstantially slower than the rate of association known to be obtainableby nucleotides in free solution. Thus, the on-rate (Kon) issubstantially and surprisingly slower than the on rate for singlenucleotides or nucleotides not attached to multivalent ligand complexes.Importantly, however, the off rate (Koff) of the multivalent ligandcomplex is substantially slower than that observed for nucleotides infree solution. Therefore, the multivalent ligand complexes of thepresent disclosure provide a surprising and beneficial improvement ofthe persistence of ternary polymerase-polynucleotide-nucleotidecomplexes (especially over such complexes that are formed with freenucleotides) allowing, for example, significant improvements in imagingquality for nucleic acid sequencing applications, over currentlyavailable methods and reagents. Importantly, this property of themultivalent substrates disclosed herein renders the formation of visibleternary complexes controllable, such that subsequent visualization,modification, or processing steps may be undertaken essentially withoutregard to the dissociation of the complex—that is, the complex can beformed, imaged, modified, or used in other ways as necessary, and willremain stable until a user carries out an affirmative dissociation step,such as exposing the complexes to a dissociation buffer.

In various embodiments, polymerases suitable for the binding interaction(e.g., during sequencing) describe herein include may include anypolymerase as is or may be known in the art. Exemplary polymerases mayinclude but are not limited to: Klenow DNA polymerase, Thermus aquaticusDNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophageT7 DNA polymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase IIIalpha and epsilon; 9 degree N polymerase, reverse transcriptases such asHIV type M or O reverse transcriptases, avian myeloblastosis virusreverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, or telomerase. Further non-limiting examples of DNApolymerases can include those from various Archaea genera, such as,Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus,Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus,Thermococcus, and Vulcanisaeta and the like or variants thereof,including such polymerases as are known in the art such as Vent™, DeepVent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases. In someembodiments, the polymerase is a Klenow polymerase.

The ternary complex has longer persistence time when the nucleotide onthe polymer-nucleotide conjugate is complementary to the target nucleicacid than when a non-complementary nucleotide. The ternary complex alsohas longer persistence time when the nucleotide on thepolymer-nucleotide conjugate is complementary to the target nucleic acidthan a complementary nucleotide that is not conjugated or tethered. Forexample, in some embodiments, said ternary complexes may have apersistence time of less than 1 s, greater than 1 s, greater than 2 s,greater than 3 s, greater than 5 s, greater than 10 s, greater than 15s, greater than 20 s, greater than 30 s, greater than 60 s, greater than120 s, greater than 360 s, greater than 3600 s, or more, or for a timelying within a range defined by any two or more of these values.

The persistence time can be measured, for example, by observing theonset and/or duration of a binding complex, such as by observing asignal from a labeled component of the binding complex. For example, alabeled nucleotide or a labeled reagent comprising one or morenucleotides may be present in a binding complex, thus allowing thesignal from the label to be detected during the persistence time of thebinding complex.

It has been observed that different ranges of persistence times areachievable with different salts or ions, showing, for example, thatcomplexes formed in the presence of, for example, magnesium form morequickly than complexes formed with other ions. It has also been observedthat complexes formed in the presence of, for example, strontium, formreadily and dissociate completely or with substantial completeness uponwithdrawal of the ion or upon washing with buffer lacking one or morecomponents of the present compositions, such as, e.g., a polymer and/orone or more nucleotides, and/or one or more interaction moieties, or abuffer containing, for example, a chelating agent which may cause oraccelerate the removal of a divalent cation from the multivalent reagentcontaining complex. Thus, in some embodiments, a composition of thepresent disclosure comprises magnesium. In some embodiments, acomposition of the present disclosure comprises calcium. In someembodiments, a composition of the present disclosure comprises strontiumor barium. In some embodiments, a composition of the present disclosurecomprises cobalt. In some embodiments, a composition of the presentdisclosure comprises MgCl₂. In some embodiments, a composition of thepresent disclosure comprises CaCl₂. In some embodiments, a compositionof the present disclosure comprises SrCl₂. In some embodiments, acomposition of the present disclosure comprises CoCl₂. In someembodiments, the composition comprises no, or substantially nomagnesium. In some embodiments, the composition comprises no, orsubstantially no calcium. In some embodiments, the methods of thepresent disclosure provide for the contacting of one or more nucleicacids with one or more of the compositions disclosed herein wherein saidcomposition lacks either one of calcium or magnesium, or lacks bothcalcium and magnesium.

The dissociation of ternary complexes can be controlled by changing thebuffer conditions. After the imaging step, a buffer with increased saltcontent is used to cause dissociation of the ternary complexes such thatlabeled polymer-nucleotide conjugates can be washed out, providing ameans by which signals can be attenuated or terminated, such as in thetransition between one sequencing cycle and the next. This dissociationmay be effected, in some embodiments, by washing the complexes with abuffer lacking a necessary metal or cofactor. In some embodiments, awash buffer may comprise one or more compositions for the purpose ofmaintaining pH control. In some embodiments, a wash buffer may compriseone or more monovalent cations, such as sodium. In some embodiments, awash buffer lacks or substantially lacks a divalent cation, for example,having no or substantially no strontium, calcium, magnesium, ormanganese. In some embodiments, a wash buffer further comprises achelating agent, such as, for example, EDTA, EGTA, nitrilotriaceticacid, polyhistidine, imidazole, or the like. In some embodiments, a washbuffer may maintain the pH of the environment at the same level as forthe bound complex. In some embodiments, a wash buffer may raise or lowerthe pH of the environment relative to the level seen for the boundcomplex. In some embodiments, the pH may be within a range from 2-4,2-7, 5-8, 7-9, 7-10, or lower than 2, or higher than 10, or a rangedefined by any two of the values provided herein.

Addition of a particular ion may affect the binding of the polymerase toa primed target nucleic acid, the formation of a ternary complex, thedissociation of a ternary complex, or the incorporation of one or morenucleotides into an elongating nucleic acid such as during a polymerasereaction. In some embodiments, relevant anions may comprise chloride,acetate, gluconate, sulfate, phosphate, or the like. In someembodiments, an ion may be included in the compositions of the presentdisclosure by the addition of one or more acids, bases, or salts, suchas NiCl₂, CoCl₂, MgCl₂, MnCl₂, SrCl₂, CaCl₂, CaSO₄, SrCO₃, BaCl₂ or thelike. Representative salts, ions, solutions and conditions may be foundin Remington: The Science and Practice of Pharmacy, 20th. Edition,Gennaro, A. R., Ed. (2000), which is hereby incorporated by reference inits entirety, and especially with respect to Chapter 17 and relateddisclosure of salts, ions, salt solutions, and ionic solutions.

The present disclosure contemplates contacting the polymer-nucleotideconjugate with one or more polymerases. The contacting can be optionallydone in the presence of one or more target nucleic acids. In someembodiments, said target nucleic acids are single stranded nucleicacids. In some embodiments, the target nucleic acids are hybridized to anucleic acid primer. In some embodiments, said target nucleic acids aredouble stranded nucleic acids. In some embodiments, said contactingcomprises contacting the polymer-nucleotide conjugate with onepolymerase. In some embodiments, said contacting comprises thecontacting of said composition comprising one or more nucleotides withmultiple polymerases. The polymerase can be bound to a single nucleicacid molecule.

The binding between target nucleic acid and polymer-nucleotide conjugatemay be provided in the presence of a polymerase that has been renderedcatalytically inactive. In one embodiment, the polymerase may have beenrendered catalytically inactive by mutation. In one embodiment, thepolymerase may have been rendered catalytically inactive by chemicalmodification. In some embodiments, the polymerase may have been renderedcatalytically inactive by the absence of a necessary substrate, ion, orcofactor. In some embodiments, the polymerase enzyme may have beenrendered catalytically inactive by the absence of magnesium ions.

The binding between target nucleic acid and polymer-nucleotide conjugateoccur in the presence of a polymerase wherein the binding solution,reaction solution, or buffer lacks a catalytic ion such as magnesium ormanganese. Alternatively, the binding between target nucleic acid andpolymer-nucleotide conjugate occur in the presence of a polymerasewherein the binding solution, reaction solution, or buffer comprises anon-catalytic ion such strontium, barium or calcium.

When the catalytically inactive polymerases are used to help a nucleicacid interact with a multivalent binding composition, the interactionbetween said composition and said polymerase stabilizes a ternarycomplex so as to render the complex detectable by fluorescence or byother methods as disclosed herein or otherwise known in the art. Unboundpolymer-nucleotide conjugates may optionally be washed away prior todetection of the ternary binding complex.

Contacting of one or more nucleic acids with the polymer-nucleotideconjugates disclosed herein in a solution containing either one ofcalcium or magnesium, or containing both calcium and magnesium.Alternatively, the contacting of one or more nucleic acids with thepolymer-nucleotide conjugates disclosed herein in a solution lackingeither one of calcium or magnesium, or lacking both calcium ormagnesium, and in a separate step, without regard to the order of thesteps, adding to the solution one of calcium or magnesium, or bothcalcium and magnesium. In some embodiments, the contacting of one ormore nucleic acids with the polymer-nucleotide conjugates disclosedherein in a solution lacking strontium or barium, and comprises in aseparate step, without regard to the order of the steps, adding to thesolution strontium.

Provided herein are methods for analyzing nucleic acids comprisingdetermining the sequence of the immobilized target nucleic acid molecule(e.g., concatemer molecule) by: (1) contacting the immobilizedconcatemer molecule with (i) a plurality of polymerases, (ii) aplurality of nucleotides, and (iii) a plurality of sequencing primersthat hybridize with the sequencing primer binding sequence, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of the immobilized concatemer molecule,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer molecule wherein the boundnucleotide incorporates into the 3′ end of the sequencing primer; (2)detecting and identifying the incorporated nucleotide therebydetermining the sequence of the immobilized concatemer molecule; and (3)optionally repeating steps (1) and (2) at least once. In someembodiments, the determining the sequence of the immobilized concatemermolecule comprises sequencing the target sequence and the spatialbarcode sequence. In some embodiments, the condition that is suitable tobind the nucleotide to the at least one of the nucleotides from theplurality to the 3′ ends of the hybridized sequencing primers andsuitable to incorporate the bound nucleotide into the hybridizedsequencing primer (step (1)) comprises at least one catalytic cationincluding magnesium and/or manganese.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (g): sequencing atleast a portion of the nucleic acid concatemer, including sequencing thetarget sequence and the spatial barcode sequence, to determine thespatial location of the target nucleic acid in the cellular biologicalsample.

In some embodiments, the sequencing of step (g) comprises sequencing atleast a portion of the nucleic acid concatemers using an optical imagingsystem comprising a field-of-view (FOV) greater than 1.0 mm². In someembodiments, the sequencing of step (g) includes placing the cellularbiological sample in a flow cell having walls (e.g., top or first wall,and bottom or second wall) and a gap in-between, where the gap can befilled with a fluid, where the flow cell is positioned in a fluorescenceoptical imaging system. The cellular biological sample has a thicknessthat may require using the imaging system to focus separately on thefirst and second surfaces of the flow cell, when using a traditionalimaging system. For improved imaging of the sequencing reaction of thenucleic acids from the cellular biological sample, the flow cell can bepositioned in a high performance fluorescence imaging system, whichcomprises two or more tube lenses which are designed to provide optimalimaging performance for the first and second surfaces of the flow cellat two or more fluorescence wavelengths. In some embodiments, thehigh-performance imaging system further comprises a focusing mechanismconfigured to refocus the optical system between acquiring images of thefirst and second surfaces of the flow cell. In some embodiments, thehigh performance imaging system is configured to image two or morefields-of-view on at least one of the first flow cell surface or thesecond flow cell surface.

In some embodiments, the sequencing of step (g) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker (FIGS. 5A and 5B).

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer (FIG. 5C), a micelle, a liposome, amicroparticle, a nanoparticle, a quantum dot, or other suitable particleknown in the art.

In some embodiments, the multivalent molecule comprises: (a) a core, and(b) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms (FIGS. 5A-D and 6A-C). In some embodiments, the spaceris attached to the linker. In some embodiments, the linker is attachedto the nucleotide unit. In some embodiments, the nucleotide unitcomprises a base, sugar and at least one phosphate group, and whereinthe linker is attached to the nucleotide unit through the base. In someembodiments, the linker comprises an aliphatic chain or an oligoethylene glycol chain where both linker chains having 2-6 subunits andoptionally the linker includes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (1) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

In Situ Single Cell Sequencing. The present disclosure provides a methodfor in situ analysis of nucleic acids in a cellular biological sample,wherein the cells of the cellular biological sample comprise cellularRNA and at least one cell in the sample having a target RNA, the methodcomprising step (a): conducting a reverse transcription reaction in thecellular biological sample under a condition that is suitable forgenerating at least one cDNA corresponding to the target RNA, whereinthe suitable condition comprises contacting the target RNA in the atleast one cell with (i) a high efficiency hybridization buffer, (ii) areverse transcriptase enzyme, (iii) a plurality of nucleotides, and (iv)a plurality of reverse transcriptase primers that bind at least aportion of the target RNA.

In some embodiments, the cellular biological sample comprises a samplethat is fresh, frozen, fresh frozen, or archived (e.g., formalin-fixedparaffin-embedded; FFPE).

In some embodiments, at least some of the target RNA remains inside thecells of the cellular biological sample. In some embodiments, the targetRNA is not immobilized to any type of support that is exterior to thecellular biological sample.

In some embodiments, the cellular biological sample is treated to fixthe location of the nucleic acids, including the target RNA, inside thecells of the sample. For example, the cellular biological sample can betreated with formalin. The cellular biological sample can be treatedwith formaldehyde, ethanol, methanol or picric acid. The cellularbiological sample can be embedded in a paraffin wax.

In some embodiments, the plurality of reverse transcriptase primers instep (a) can be modified so they bind to cells or bind to cellularcomponents in a cell, such that the cDNA generated by conducting thereverse transcriptase reaction binds a cellular component and remains inthe cell. For example, the reverse transcriptase primers can be modifiedto include a reactive moiety at their 5′ ends or can include nucleotideresidues that are modified to include a reactive moiety. The reactivemoiety comprise nucleophilic functional groups (e.g., amines, alcohols,thiols and hydrazides), electrophilic functional groups (e.g.,aldehydes, esters, epoxides, isocyanates, maleimides and vinyl ketones),functional groups capable of cycloaddition reactions, forming disulfidebonds, or binding to metals. The reactive moiety comprises primary orsecondary amines, lower alkylamine group, acetyl group, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,maleimides, oxycarbonylimidazoles, nitrophenylesters, trifluoroethylesters, glycidyl ethers or vinylsulfones. The reactive moiety comprisesan affinity binding group such as biotin. The reactive moiety comprisesfluorescein or acridine.

In some embodiments, the reverse transcription reaction of step (a)comprises a plurality of nucleotides and an enzyme having reversetranscription activity, including reverse transcriptase enzymes from AMV(avian myeloblastosis virus), M-MLV (moloney murine leukemia virus), orHIV (human immunodeficiency virus). In some embodiments, the reversetranscriptase can be a commercially-available enzyme, includingMultiScribe™ ThermoScript™, or ArrayScript™. In some embodiments, thereverse transcriptase enzyme comprises Superscript I, II, III, or IVenzymes. In some embodiments, the reverse transcription reaction caninclude an RNase inhibitor. In some embodiments, the plurality ofreverse transcription primers are resistant to ribonuclease degradation.For example, the reverse transcription primers can be modified toinclude two or more phosphorothioate bonds, or 2′-O-methyl, 2′fluoro-bases, phosphorylated 3′ ends, or locked nucleic acid residues.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (a) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiency highefficiency hybridization buffer of step (a) comprises: (i) the firstpolar aprotic solvent comprises acetonitrile at 25-50% by volume of thehigh efficiency high efficiency hybridization buffer; (ii) the secondpolar aprotic solvent comprises formamide at 5-10% by volume of the highefficiency high efficiency hybridization buffer; (iii) the pH buffersystem comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG)at 5-35% by volume of the high efficiency high efficiency hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (a) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

In some embodiments, the method for in situ analysis of nucleic acids ina cellular biological sample further comprises step (b): degrading someor all of the cellular RNA and retaining at least the cell membrane ofthe cellular biological sample. In some embodiment, the cellular RNA isdegraded with a ribonuclease.

In some embodiments, the method for in situ analysis of nucleic acids ina cellular biological sample further comprises step (c): contacting theat least one cDNA with a plurality of padlock probes each comprising twoterminal regions that bind to portions of the at least one cDNA togenerate at least one cDNA-padlock probe complex having the two probeterminal regions hybridized to the adjacent regions of the cDNA to forma nick or gap.

In some embodiments, the padlock probe of step (c) comprises a singleoligonucleotide strand which includes target capture sequences at its 5′terminal-end and 3′ terminal-end that are complementary to contiguousregions of the target nucleic acid molecule (e.g., RNA). The padlockprobe can also include any one or any combination of two or more adaptorsequences including an amplification primer binding sequence, asequencing primer binding sequence, an immobilization sequence and/or asample index sequence. The various adaptor sequences can be located inany region, for example the internal portion of the padlock probe. The5′ and 3′ ends of the padlock probe can hybridize to adjacent positionson the target nucleic acid molecule to form an open circularizedmolecule with a nick or gap between the hybridized 5′ and 3′ ends.

In some embodiments, the method for in situ analysis of nucleic acids ina cellular biological sample further comprises step (d): conducting agap-filling reaction and/or a ligation reaction on the at least onecDNA-padlock probe complex to generate a covalently closed circularizedpadlock probe.

In some embodiments, the gap-filling reaction comprises contacting theopen circularized molecule with a DNA polymerase and a plurality ofnucleotides, where the DNA polymerase comprises E. coli DNA polymeraseI, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4DNA polymerase. In some embodiments, the ligation reaction comprises useof a ligase enzyme, including a T3, T4, T7 or Taq DNA ligase enzyme.

In some embodiments, the method for in situ analysis of nucleic acids ina cellular biological sample further comprises step (e): conducting arolling circle amplification reaction on the circularized padlock probesto generate a plurality of nucleic acid concatemers.

In some embodiments, the rolling circle amplification reaction of step(e) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(e) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(e) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C., or from room temperature to about65° C.

In some embodiments, the rolling circle amplification reaction of step(e) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(e) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiments, the method for in situ analysis of nucleic acids ina cellular biological sample further comprises step (f): sequencing atleast a portion of the nucleic acid concatemers. In some embodiments,the sequencing comprises sequencing at least a portion of the nucleicacid concatemers using an optical imaging system comprising afield-of-view (FOV) greater than 1.0 mm².

In some embodiments, the sequencing of step (f) includes placing thecellular biological sample in a flow cell having walls (e.g., top orfirst wall, and bottom or second wall) and a gap in-between, where thegap can be filled with a fluid, where the flow cell is positioned in afluorescence optical imaging system. The cellular biological sample hasa thickness that may require using the imaging system to focusseparately on the first and second surfaces of the flow cell, when usinga traditional imaging system. For improved imaging of the sequencingreaction in the cellular biological sample, the flow cell can bepositioned in a high performance fluorescence imaging system, whichcomprises two or more tube lenses which are designed to provide optimalimaging performance for the first and second surfaces of the flow cellat two or more fluorescence wavelengths. In some embodiments, thehigh-performance imaging system further comprises a focusing mechanismconfigured to refocus the optical system between acquiring images of thefirst and second surfaces of the flow cell. In some embodiments, thehigh performance imaging system is configured to image two or morefields-of-view on at least one of the first flow cell surface or thesecond flow cell surface.

In some embodiments, steps (a)-(f) are conducted inside the cellularbiological sample. In some embodiments, the cellular biological sampleis positioned on a support prior to step (a), where the support lacksimmobilized capture oligonucleotides. In some embodiments, the targetRNA or cDNA is not immobilized to any type of support. In someembodiments, at least some of the target RNA and/or cDNA remains insidethe cellular biological sample throughout steps (a)-(f).

In some embodiments, the sequencing of step (f) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker.

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

In some embodiments, the multivalent molecule comprises: (1) a core, and(2) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms. In some embodiments, the spacer is attached to thelinker. In some embodiments, the linker is attached to the nucleotideunit. In some embodiments, the nucleotide unit comprises a base, sugarand at least one phosphate group, and wherein the linker is attached tothe nucleotide unit through the base. In some embodiments, the linkercomprises an aliphatic chain or an oligo ethylene glycol chain whereboth linker chains having 2-6 subunits and optionally the linkerincludes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (f) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (1) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (f) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

In situ Single Cell Sequencing. The present disclosure provides a methodfor in situ analysis of nucleic acids in a single cell wherein thesingle cell is placed in a cell media, and wherein the single cellcomprises cellular RNA including a target RNA, the method comprising:(a) conducting a reverse transcription reaction in the single cell undera condition that is suitable for generating at least one cDNAcorresponding to the target RNA, wherein the suitable conditioncomprises contacting the target RNA in the single cell with (i) a highefficiency hybridization buffer, (ii) a reverse transcriptase enzyme,(iii) a plurality of nucleotides, and (iv) a plurality of reversetranscriptase primers that bind at least a portion of the target RNA.

In some embodiments, the single cell is a cell sample that is fresh,frozen, fresh frozen, or archived (e.g., formalin-fixedparaffin-embedded; FFPE).

In some embodiments, the target RNA remains inside the single cell. Insome embodiments, the target RNA is not immobilized to any type ofsupport that is exterior to the single cell.

In some embodiments, the single cell can be treated to fix the locationof the nucleic acids, including the target RNA, inside the single cell.For example, the single cell can be treated with formalin. The singlecell can be treated with formaldehyde, ethanol, methanol or picric acid.The single cell can be embedded in a paraffin wax.

In some embodiments, the plurality of reverse transcriptase primers instep (a) can be modified so they bind to cells or bind to cellularcomponents in a cell, such that the cDNA generated by conducting thereverse transcriptase reaction binds a cellular component and remains inthe cell. For example, the reverse transcriptase primers can be modifiedto include a reactive moiety at their 5′ ends or can include nucleotideresidues that are modified to include a reactive moiety. The reactivemoiety comprise nucleophilic functional groups (e.g., amines, alcohols,thiols and hydrazides), electrophilic functional groups (e.g.,aldehydes, esters, epoxides, isocyanates, maleimides and vinyl ketones),functional groups capable of cycloaddition reactions, forming disulfidebonds, or binding to metals. The reactive moiety comprises primary orsecondary amines, lower alkylamine group, acetyl group, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,maleimides, oxycarbonylimidazoles, nitrophenylesters, trifluoroethylesters, glycidyl ethers or vinylsulfones. The reactive moiety comprisesan affinity binding group such as biotin. The reactive moiety comprisesfluorescein or acridine.

In some embodiments, the reverse transcription reaction of step (a)comprises a plurality of nucleotides and an enzyme having reversetranscription activity, including reverse transcriptase enzymes from AMV(avian myeloblastosis virus), M-MLV (moloney murine leukemia virus), orHIV (human immunodeficiency virus). In some embodiments, the reversetranscriptase can be a commercially-available enzyme, includingMultiScribe™ ThermoScript™, or ArrayScript™. In some embodiments, thereverse transcriptase enzyme comprises Superscript I, II, III, or IVenzymes. In some embodiments, the reverse transcription reaction caninclude an RNase inhibitor. In some embodiments, the plurality ofreverse transcription primers are resistant to ribonuclease degradation.For example, the reverse transcription primers can be modified toinclude two or more phosphorothioate bonds, or 2′-O-methyl, 2′fluoro-bases, phosphorylated 3′ ends, or locked nucleic acid residues.

In some embodiments, the plurality of reverse transcription primers areresistant to ribonuclease degradation. For example, the reversetranscription primers can be modified to include two or morephosphorothioate bonds, or 2′-O-methyl, 2′ fluoro-bases, phosphorylated3′ ends, or locked nucleic acid residues.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (a) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiency highefficiency hybridization buffer of step (a) comprises: (i) the firstpolar aprotic solvent comprises acetonitrile at 25-50% by volume of thehigh efficiency high efficiency hybridization buffer; (ii) the secondpolar aprotic solvent comprises formamide at 5-10% by volume of the highefficiency high efficiency hybridization buffer; (iii) the pH buffersystem comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG)at 5-35% by volume of the high efficiency high efficiency hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (a) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

In some embodiments, the single cell is placed in a cell media whichcomprises a complex cell media having a fluid obtained from a biologicalfluid which is selected from a group consisting of fetal bovine serum,blood plasma, blood serum, lymph fluid, human placental cord serum andamniotic fluid, and wherein the complex cell media can support cellgrowth and/or proliferation. In some embodiments, the complex cell mediacomprises a serum-containing media, a serum-free media, achemically-defined media, or a protein-free media. In some embodiments,the complex cell media comprises RPMI-1640, MEM, DMEM or IMDM.

In some embodiments, the single cell is placed in a cell media whichcomprises a simple cell media which includes any one or any combinationof two or more of a buffer, a phosphate compound, a sodium compound, apotassium compound, a calcium compound, a magnesium compound and/orglucose, and wherein the simple cell media cannot support cell growthand/or proliferation. In some embodiments, the simple cell mediacomprise PBS, DPBS, HBSS, DMEM, EMEM or EBSS.

In some embodiments, the method for in situ analysis of nucleic acids ina single cell further comprise step (b): degrading some or all of thecellular RNA and retaining at least the cell membrane of the singlecell. In some embodiment, the cellular RNA is degraded with aribonuclease.

In some embodiments, the method for in situ analysis of nucleic acids ina single cell further comprise step (c): contacting the at least onecDNA with a plurality of padlock probes each comprising two terminalregions that bind to portions of the at least one cDNA to generate atleast one cDNA-padlock probe complex having the two probe terminalregions hybridized to the adjacent regions of the cDNA to form a nick orgap.

In some embodiments, the padlock probe of step (c) comprises a singleoligonucleotide strand which includes target capture sequences at its 5′terminal-end and 3′ terminal-end that are complementary to contiguousregions of the target nucleic acid molecule (e.g., RNA). The padlockprobe can also include any one or any combination of two or more adaptorsequences including an amplification primer binding sequence, asequencing primer binding sequence, an immobilization sequence and/or asample index sequence. The various adaptor sequences can be located inany region, for example the internal portion of the padlock probe. The5′ and 3′ ends of the padlock probe can hybridize to adjacent positionson the target nucleic acid molecule to form an open circularizedmolecule with a nick or gap between the hybridized 5′ and 3′ ends.

In some embodiments, the method for in situ analysis of nucleic acids ina single cell further comprise step (d): conducting a gap-fillingreaction and/or a ligation reaction on the at least one cDNA-padlockprobe complex to generate a covalently closed circularized padlockprobe.

In some embodiments, the gap-filling reaction comprises contacting theopen circularized molecule with a DNA polymerase and a plurality ofnucleotides, where the DNA polymerase comprises E. coli DNA polymeraseI, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4DNA polymerase. In some embodiments, the ligation reaction comprises useof a ligase enzyme, including a T3, T4, T7 or Taq DNA ligase enzyme.

In some embodiments, the method for in situ analysis of nucleic acids ina single cell further comprise step (e): conducting a rolling circleamplification reaction on the covalently closed circularized padlockprobes to generate a plurality of nucleic acid concatemers.

In some embodiments, the rolling circle amplification reaction of step(e) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(e) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(e) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C., or from room temperature to about65° C.

In some embodiments, the rolling circle amplification reaction of step(e) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(e) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiments, the method for in situ analysis of nucleic acids ina single cell further comprise step (I): sequencing at least a portionof the nucleic acid concatemers. In some embodiments, the sequencingcomprises sequencing at least a portion of the nucleic acid concatemersusing an optical imaging system comprising a field-of-view (FOV) greaterthan 1.0 mm².

In some embodiments, the sequencing of step (f) includes placing thesingle cell in a flow cell having walls (e.g., top or first wall, andbottom or second wall) and a gap in-between, where the gap can be filledwith a fluid, where the flow cell is positioned in a fluorescenceoptical imaging system. The single cell has a thickness that may requireusing the imaging system to focus separately on the first and secondsurfaces of the flow cell, when using a traditional imaging system. Forimproved imaging of the sequencing reaction in the single cell, the flowcell can be positioned in a high performance fluorescence imagingsystem, which comprises two or more tube lenses which are designed toprovide optimal imaging performance for the first and second surfaces ofthe flow cell at two or more fluorescence wavelengths. In someembodiments, the high-performance imaging system further comprises afocusing mechanism configured to refocus the optical system betweenacquiring images of the first and second surfaces of the flow cell. Insome embodiments, the high performance imaging system is configured toimage two or more fields-of-view on at least one of the first flow cellsurface or the second flow cell surface.

In some embodiments, steps (a)-(f) are conducted inside the single cell.In some embodiments, the target RNA or cDNA is not immobilized to anytype of support. In some embodiments, at least some of the target RNAand/or cDNA remains inside the cellular biological sample throughoutsteps (a)-(f).

In some embodiments, the single cell is positioned on a support prior toany of steps (a)-(f), where the support lacks immobilized captureoligonucleotides. For example, the method comprises: (1) positioning thesingle cell on a low non-specific binding coating that lacks immobilizedcapture oligonucleotides under a condition suitable for immobilizing thesingle cell to the surface of the low non-specific binding support,wherein the positioning is conducted prior to step (a), and wherein thecellular RNA remains inside the single cell; (2) positioning the singlecell on a low non-specific binding coating that lacks immobilizedcapture oligonucleotides under a condition suitable for immobilizing thesingle cell to the surface of the low non-specific binding support,wherein the positioning is conducted prior to step (b), and wherein theat least one cDNA remains inside the single cell; (3) positioning thesingle cell on a low non-specific binding coating that lacks immobilizedcapture oligonucleotides under a condition suitable for immobilizing thesingle cell to the surface of the low non-specific binding support,wherein the positioning is conducted prior to step (e), and wherein thecircularized padlock probe remains inside the single cell; or (4)positioning the single cell on a low non-specific binding coating thatlacks immobilized capture oligonucleotides under a condition suitablefor immobilizing the single cell to the surface of the low non-specificbinding support, wherein the positioning is conducted prior to step (f),and wherein the plurality of nucleic acid concatemers remain inside thesingle cell.

In some embodiments, the low non-specific binding support comprises asupport with a coating, wherein the coating comprises at least onehydrophilic polymer layer having a water contact angle of no more than45 degrees.

In some embodiments, the sequencing of step (f) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker.

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

In some embodiments, the multivalent molecule comprises: (1) a core, and(2) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms. In some embodiments, the spacer is attached to thelinker. In some embodiments, the linker is attached to the nucleotideunit. In some embodiments, the nucleotide unit comprises a base, sugarand at least one phosphate group, and wherein the linker is attached tothe nucleotide unit through the base. In some embodiments, the linkercomprises an aliphatic chain or an oligo ethylene glycol chain whereboth linker chains having 2-6 subunits and optionally the linkerincludes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (f) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (i) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (f) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

Biological Molecule Capture on a Low Binding Coating and Analysis. Thepresent disclosure provides method for analyzing biological moleculesfrom a cellular biological sample, wherein the cells in the cellularbiological sample comprise cellular nucleic acids and polypeptides, andwherein at least one cell in the sample includes a target nucleic acidthat encodes a target polypeptide, the method comprising the generalstep of: (a) providing a support comprising a low non-specific bindingcoating to which a plurality of capture oligonucleotides and optionallya plurality of circularization oligonucleotides are immobilized, whereinthe plurality of immobilized capture oligonucleotides comprise (i) atarget capture region that hybridizes to at least a portion of a targetnucleic acid molecule, and (ii) a spatial barcode sequence, wherein thelow non-specific binding coating comprises at least one hydrophilicpolymer layer having a water contact angle of no more than 45 degrees.

In some embodiments, the low non-specific binding coating in step (a)exhibits low background fluorescence signals or high contrast to noise(CNR) ratios relative to known surfaces in the art. In some embodiments,the low non-specific binding coating exhibits a level of non-specificCy3 dye absorption of less than about 0.25 molecules/μm², where no morethan 5% of the target nucleic acid is associated with the surfacecoating without hybridizing to an immobilized capture oligonucleotide.In some embodiments, a fluorescence image of the surface coating havinga plurality of clonally-amplified clusters of nucleic acid exhibits acontrast-to-noise ratio (CNR) of at least 20, or at least 50, or highercontrast-to-noise ratios (CNR), when using a fluorescence imaging systemunder non-signal saturating conditions.

In some embodiments, the low non-specific binding coating of step (a)has regions (e.g., features) located at pre-determined locations on thecoating. The low non-specific binding coating comprises a plurality offeatures including at least a first and second feature, where eachfeature includes a plurality of capture oligonucleotide and optionally aplurality of circularization oligonucleotides that are immobilized tothe coating. In some embodiments, the first feature comprises aplurality of first capture oligonucleotides having a first targetcapture region and a first spatial barcode sequence. In someembodiments, the second feature comprises a plurality of second captureoligonucleotides having a second target capture region and a secondspatial barcode sequence. In some embodiments, the sequence of the firsttarget capture region in the first feature is the same or different fromthe sequence of the second target capture region in the second feature.In some embodiments, the first spatial barcode sequence in the firstfeature differs from the second spatial barcode sequence in the secondfeature.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (b): contacting thelow non-specific binding coating with the cellular biological sample inthe presence of a high efficiency hybridization buffer under conditionssuitable to promote migration of the cellular nucleic acids, includingthe target nucleic acid molecule, from the cellular biological sample toone of the immobilized capture oligonucleotides thereby forming animmobilized target nucleic acid duplex, wherein the target nucleic acidmolecule is immobilized to the low non-specific binding coating in amanner that preserves spatial location information of the target nucleicacid molecule in the cellular biological sample.

In some embodiments, the cellular biological sample in step (b)comprises a cellular biological sample that is fresh, frozen, freshfrozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE).

In some embodiments, the cellular biological sample in step (b) issubjected to a permeabilizing reaction to promote migration of thecellular nucleic acid molecules (e.g., DNA and/or RNA), including thetarget nucleic acid molecule, from the cellular biological sample to oneof the immobilized capture oligonucleotides.

In some embodiments, the target nucleic acid comprises RNA. In someembodiments, the spatial location of the target RNA in the cellularbiological sample corresponds to the spatial location of at least onecell in the cellular biological sample that expresses the target RNAwhich encodes the target polypeptide.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the high efficiency highefficiency hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the high efficiency highefficiency hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe high efficiency high efficiency hybridization buffer. In someembodiments, the high efficiency hybridization buffer further comprisesbetaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (c): conducting aprimer extension reaction on the immobilized target nucleic acid duplexthereby forming an immobilized target extension product.

In some embodiments, the primer extension reaction of step (c) can be areverse transcription reaction which comprises (i) a reversetranscriptase enzyme, (ii) a plurality of nucleotides, and (iii) aplurality of reverse transcriptase primers that bind at least a portionof the target RNA. In some embodiments, the reverse transcriptionreaction of step (a) comprises a plurality of nucleotides and an enzymehaving reverse transcription activity, including reverse transcriptaseenzymes from AMV (avian myeloblastosis virus), M-MLV (moloney murineleukemia virus), or HIV (human immunodeficiency virus). In someembodiments, the reverse transcriptase can be a commercially-availableenzyme, including MultiScribe™ ThermoScript™, or ArrayScript™. In someembodiments, the reverse transcriptase enzyme comprises Superscript I,II, III, or IV enzymes. In some embodiments, the reverse transcriptionreaction can include an RNase inhibitor.

In some embodiments, the plurality of reverse transcription primers areresistant to ribonuclease degradation. For example, the reversetranscription primers can be modified to include two or morephosphorothioate bonds, or 2′-O-methyl, 2′ fluoro-bases, phosphorylated3′ ends, or locked nucleic acid residues.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (d): forming an opencircular target molecule using the immobilized circularizationoligonucleotide, or if the low non-specific binding coating does notalready include an immobilized circularization oligonucleotide thenimmobilizing a soluble circularization oligonucleotide to the lownon-specific binding coating in proximity to the immobilized targetextension product and forming an open circular target molecule using thenow-immobilized circularization oligonucleotide;

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (e): forming acovalently closed circular target molecule which is immobilized to thelow non-specific binding coating.

In some embodiments, the forming the covalently closed circular targetmolecule comprises a polymerase-mediated gap-filling reaction, anenzymatic ligation reaction, or a polymerase-mediated gap-fillingreaction and enzymatic ligation reaction. In some embodiments, thepolymerase-mediate gap-filling reaction comprises contacting the opencircular target molecule with a DNA polymerase and a plurality ofnucleotides, where the DNA polymerase comprises E. coli DNA polymeraseI, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4DNA polymerase. In some embodiments, the enzymatic ligation reactioncomprises use of a ligase enzyme, including a T3, T4, T7 or Taq DNAligase enzyme. In some embodiments, the forming the covalently closedcircular target molecule comprises contacting the open circular targetmolecule with a CircLigase or CircLigase II enzyme.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (f): conducting arolling circle amplification reaction on the immobilized covalentlyclosed circular target molecule to form an immobilized nucleic acidconcatemer molecule having tandem repeat regions comprising the targetsequence and the spatial barcode sequence.

In some embodiments, the rolling circle amplification reaction of step(f) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(f) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(f) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C., or from room temperature to about65° C.

In some embodiments, the rolling circle amplification reaction of step(f) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(f) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiment, the rolling circle amplification reaction can befollowed by a flexing amplification reaction instead of a multipledisplacement amplification (MDA) reaction. In some embodiments, theflexing amplification reaction comprises: (1) forming a nucleic acidrelaxant reaction mixture by contacting the nucleic acid concatemer withone or a combination of two or more compounds selected from a groupconsisting of formamide, acetonitrile, ethanol, guanidine hydrochloride,urea, potassium iodide and/or polyamines, to generate a relaxed nucleicacid concatemer, wherein the forming a nucleic acid relaxant reactionmixture is conducted with a temperature ramp-up, a relaxant incubationtemperature, and a temperature ramp-down; (2) washing the relaxedconcatemer; (3) forming a flexing amplification reaction mixture bycontacting the relaxed concatemer with a strand-displacing DNApolymerase, a plurality of nucleotides, a catalytic divalent cation, (inthe absence of added amplification primers), to generate double-strandedconcatemers, wherein the forming a flexing amplification reactionmixture is conducted with a temperature ramp-up, a flexing incubationtemperature, and a temperature ramp-down; (4) washing thedouble-stranded concatemer; and (5) repeating steps (1)-(4) at leastonce.

In some embodiments, the method for analyzing biological molecules froma cellular biological sample further comprise step (g): sequencing atleast a portion of the nucleic acid concatemer, including sequencing thetarget sequence and the spatial barcode sequence, to determine thespatial location of the target nucleic acid in the cellular biologicalsample.

In some embodiments, the sequencing of step (g) comprises sequencing atleast a portion of the nucleic acid concatemers using an optical imagingsystem comprising a field-of-view (FOV) greater than 1.0 mm². In someembodiments, the sequencing of step (g) includes placing the cellularbiological sample in a flow cell having walls (e.g., top or first wall,and bottom or second wall) and a gap in-between, where the gap can befilled with a fluid, where the flow cell is positioned in a fluorescenceoptical imaging system. The cellular biological sample has a thicknessthat may require using the imaging system to focus separately on thefirst and second surfaces of the flow cell, when using a traditionalimaging system. For improved imaging of the sequencing reaction of thenucleic acids from the cellular biological sample, the flow cell can bepositioned in a high performance fluorescence imaging system, whichcomprises two or more tube lenses which are designed to provide optimalimaging performance for the first and second surfaces of the flow cellat two or more fluorescence wavelengths. In some embodiments, thehigh-performance imaging system further comprises a focusing mechanismconfigured to refocus the optical system between acquiring images of thefirst and second surfaces of the flow cell. In some embodiments, thehigh performance imaging system is configured to image two or morefields-of-view on at least one of the first flow cell surface or thesecond flow cell surface.

In some embodiments, the sequencing of step (g) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker.

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

In some embodiments, the multivalent molecule comprises: (1) a core, and(2) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms. In some embodiments, the spacer is attached to thelinker. In some embodiments, the linker is attached to the nucleotideunit. In some embodiments, the nucleotide unit comprises a base, sugarand at least one phosphate group, and wherein the linker is attached tothe nucleotide unit through the base. In some embodiments, the linkercomprises an aliphatic chain or an oligo ethylene glycol chain whereboth linker chains having 2-6 subunits and optionally the linkerincludes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (i) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

Capturing Nucleic Acids from A Single Cell and Analysis. The presentdisclosure provides a method for analyzing nucleic acids from a singlecell (e.g., a cellular biological sample) wherein the single cell isplaced in a cell media, and wherein the single cell includes cellularnucleic acids and polypeptides, and wherein the single cell includes atarget nucleic acid that encodes a target polypeptide, the methodcomprising the general steps of: (a) providing a support comprising alow non-specific binding coating to which a plurality of captureoligonucleotides and optionally a plurality of circularizationoligonucleotides are immobilized, wherein the plurality of immobilizedcapture oligonucleotides comprise (i) a target capture region thathybridizes to at least a portion of a target nucleic acid molecule, and(ii) a spatial barcode sequence, wherein the low non-specific bindingcoating comprises at least one hydrophilic polymer layer having a watercontact angle of no more than 45 degrees.

In some embodiments, the low non-specific binding coating in step (a)exhibits low background fluorescence signals or high contrast to noise(CNR) ratios relative to known surfaces in the art. In some embodiments,the low non-specific binding coating exhibits a level of non-specificCy3 dye absorption of less than about 0.25 molecules/μm², where no morethan 5% of the target nucleic acid is associated with the surfacecoating without hybridizing to an immobilized capture oligonucleotide.In some embodiments, a fluorescence image of the surface coating havinga plurality of clonally-amplified clusters of nucleic acid exhibits acontrast-to-noise ratio (CNR) of at least 20, or at least 50, or highercontrast-to-noise ratios (CNR), when using a fluorescence imaging systemunder non-signal saturating conditions.

In some embodiments, the low non-specific binding coating of step (a)has regions (e.g., features) located at pre-determined locations on thecoating. The low non-specific binding coating comprises a plurality offeatures including at least a first and second feature, where eachfeature includes a plurality of capture oligonucleotide and optionally aplurality of circularization oligonucleotides that are immobilized tothe coating. In some embodiments, the first feature comprises aplurality of first capture oligonucleotides having a first targetcapture region and a first spatial barcode sequence. In someembodiments, the second feature comprises a plurality of second captureoligonucleotides having a second target capture region and a secondspatial barcode sequence. In some embodiments, the sequence of the firsttarget capture region in the first feature is the same or different fromthe sequence of the second target capture region in the second feature.In some embodiments, the first spatial barcode sequence in the firstfeature differs from the second spatial barcode sequence in the secondfeature.

In some embodiments, the single cell is placed in a cell media whichcomprises a complex cell media having a fluid obtained from a biologicalfluid which is selected from a group consisting of fetal bovine serum,blood plasma, blood serum, lymph fluid, human placental cord serum andamniotic fluid, and wherein the complex cell media can support cellgrowth and/or proliferation. In some embodiments, the complex cell mediacomprises a serum-containing media, a serum-free media, achemically-defined media, or a protein-free media. In some embodiments,the complex cell media comprises RPMI-1640, MEM, DMEM or IMDM.

In some embodiments, the single cell is placed in a cell media whichcomprises a simple cell media which includes any one or any combinationof two or more of a buffer, a phosphate compound, a sodium compound, apotassium compound, a calcium compound, a magnesium compound and/orglucose, and wherein the simple cell media cannot support cell growthand/or proliferation. In some embodiments, the simple cell mediacomprise PBS, DPBS, HBSS, DMEM, EMEM or EBSS.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (b): contacting the lownon-specific binding coating with the single cell in the presence of ahigh efficiency hybridization buffer under conditions suitable topromote migration of the cellular nucleic acids, including the targetnucleic acid molecule, from the single cell to one of the immobilizedcapture oligonucleotides thereby forming an immobilized target nucleicacid duplex, wherein the target nucleic acid molecule from the singlecell is immobilized to the low non-specific binding coating in a mannerthat preserves spatial location information of the target nucleic acidmolecule in the single cell.

In some embodiments, the single cell in step (b) comprises a single cellsample that is fresh, frozen, fresh frozen, or archived (e.g.,formalin-fixed paraffin-embedded; FFPE).

In some embodiments, the single cell in step (b) is subjected to apermeabilizing reaction to promote migration of the cellular nucleicacid molecules (e.g., DNA and/or RNA), including the target nucleic acidmolecule, from the single cell to one of the immobilized captureoligonucleotides.

In some embodiments, the target nucleic acid comprises RNA. In someembodiments, the spatial location of the target RNA in the single cellcorresponds to the spatial location of the target RNA which encodes thetarget polypeptide.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) a first polar aprotic solvent having adielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the highefficiency high efficiency hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the high efficiency high efficiencyhybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the high efficiency highefficiency hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the high efficiency highefficiency hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe high efficiency high efficiency hybridization buffer. In someembodiments, the high efficiency hybridization buffer further comprisesbetaine.

In some embodiments, the high efficiency high efficiency hybridizationbuffer of step (b) promotes high stringency (e.g., specificity), speed,and efficacy of nucleic acid hybridization reactions and increases theefficiency of the subsequent amplification and sequencing steps. In someembodiments, the high efficiency hybridization buffer significantlyshortens nucleic acid hybridization times, and decreases sample inputrequirements. Nucleic acid annealing can be performed at isothermalconditions and eliminate the cooling step for annealing.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (c): conducting a primer extensionreaction on the immobilized target nucleic acid duplex thereby formingan immobilized target extension product.

In some embodiments, the primer extension reaction of step (c) can be areverse transcription reaction which comprises (i) a reversetranscriptase enzyme, (ii) a plurality of nucleotides, and (iii) aplurality of reverse transcriptase primers that bind at least a portionof the target RNA. In some embodiments, the reverse transcriptionreaction of step (a) comprises a plurality of nucleotides and an enzymehaving reverse transcription activity, including reverse transcriptaseenzymes from AMV (avian myeloblastosis virus), M-MLV (moloney murineleukemia virus), or HIV (human immunodeficiency virus). In someembodiments, the reverse transcriptase can be a commercially-availableenzyme, including MultiScribe™ ThermoScript™, or ArrayScript™. In someembodiments, the reverse transcriptase enzyme comprises Superscript I,II, III, or IV enzymes. In some embodiments, the reverse transcriptionreaction can include an RNase inhibitor.

In some embodiments, the plurality of reverse transcription primers areresistant to ribonuclease degradation. For example, the reversetranscription primers can be modified to include two or morephosphorothioate bonds, or 2′-O-methyl, 2′ fluoro-bases, phosphorylated3′ ends, or locked nucleic acid residues.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (d): forming an open circulartarget molecule using the immobilized circularization oligonucleotide,or if the low non-specific binding coating does not already include animmobilized circularization oligonucleotide then immobilizing a solublecircularization oligonucleotide to the low non-specific binding coatingin proximity to the immobilized target extension product and forming anopen circular target molecule using the now-immobilized circularizationoligonucleotide.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (e): forming a covalently closedcircular target molecule which is immobilized to the low non-specificbinding coating.

In some embodiments, the forming the covalently closed circular targetmolecule comprises a polymerase-mediated gap-filling reaction, anenzymatic ligation reaction, or a polymerase-mediated gap-fillingreaction and enzymatic ligation reaction. In some embodiments, thepolymerase-mediate gap-filling reaction comprises contacting the opencircular target molecule with a DNA polymerase and a plurality ofnucleotides, where the DNA polymerase comprises E. coli DNA polymeraseI, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4DNA polymerase. In some embodiments, the enzymatic ligation reactioncomprises use of a ligase enzyme, including a T3, T4, T7 or Taq DNAligase enzyme. In some embodiments, the forming the covalently closedcircular target molecule comprises contacting the open circular targetmolecule with a CircLigase or CircLigase II enzyme.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (f): conducting a rolling circleamplification reaction on the immobilized covalently closed circulartarget molecule to form an immobilized nucleic acid concatemer moleculehaving tandem repeat regions comprising the target sequence and thespatial barcode sequence.

In some embodiments, the rolling circle amplification reaction of step(f) comprises contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one catalytic divalent cation, under a condition suitable forgenerating at least one nucleic acid concatemer, wherein the at leastone catalytic divalent cation comprises magnesium or manganese.

In some embodiments, the rolling circle amplification reaction of step(f) comprises: (1) contacting the covalently closed circularized padlockprobes (e.g., circularized nucleic acid template molecule(s)) with anamplification primer, a DNA polymerase, a plurality of nucleotides, andat least one non-catalytic divalent cation that does not promotepolymerase-catalyzed nucleotide incorporation into the amplificationprimer, wherein the non-catalytic divalent cation comprises strontium orbarium; and (2) contacting the covalently closed circularized padlockprobes with at least one catalytic divalent cation, under a conditionsuitable for generating at least one nucleic acid concatemer, whereinthe at least one catalytic divalent cation comprises magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction of step(f) is conducted at a constant temperature (e.g., isothermal) rangingfrom room temperature to about 50° C.

In some embodiments, the rolling circle amplification reaction of step(f) can be conducted in the presence of a plurality of compactionoligonucleotides which compacts the size and/or shape of the immobilizedconcatemer to form an immobilized compact nanoball.

In some embodiments, the rolling circle amplification reaction of step(f) comprises a DNA polymerase having a strand displacing activity whichis selected from a group consisting of phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase,and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. In some embodiments,the phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), and chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith at least one amplification primer comprising a random sequence, aDNA polymerase having strand displacement activity, a plurality ofnucleotides, and a catalytic divalent cation comprising magnesium ormanganese.

In some embodiments, the rolling circle amplification reaction can befollowed by a multiple displacement amplification (MDA) reaction. Insome embodiments, the method further comprises: conducting a multipledisplacement amplification (MDA) reaction prior to step (f), wherein theMDA reaction comprises contacting at least one nucleic acid concatemerwith a DNA primase-polymerase enzyme, a DNA polymerase having stranddisplacement activity, a plurality of nucleotides, and a catalyticdivalent cation comprising magnesium or manganese. In some embodiments,a DNA primase-polymerase comprises an enzyme having activities of a DNApolymerase and an RNA primase. A DNA primase-polymerase enzyme canutilize deoxyribonucleotide triphosphates to synthesize a DNA primer ona single-stranded DNA template in a template-sequence dependent manner,and can extend the primer strand via nucleotide polymerization (e.g.,primer extension), in the presence of a catalytic divalent cation (e.g.,magnesium and/or manganese). The DNA primase-polymerase include enzymesthat are members of DnaG-like primases (e.g., bacteria) and AEP-likeprimases (Archaea and Eukaryotes). An exemplary DNA primase-polymeraseenzyme is Tth PrimPol from Thermus thermophilus HB27.

In some embodiment, the rolling circle amplification reaction can befollowed by a flexing amplification reaction instead of a multipledisplacement amplification (MDA) reaction. In some embodiments, theflexing amplification reaction comprises: (1) forming a nucleic acidrelaxant reaction mixture by contacting the nucleic acid concatemer withone or a combination of two or more compounds selected from a groupconsisting of formamide, acetonitrile, ethanol, guanidine hydrochloride,urea, potassium iodide and/or polyamines, to generate a relaxed nucleicacid concatemer, wherein the forming a nucleic acid relaxant reactionmixture is conducted with a temperature ramp-up, a relaxant incubationtemperature, and a temperature ramp-down; (2) washing the relaxedconcatemer; (3) forming a flexing amplification reaction mixture bycontacting the relaxed concatemer with a strand-displacing DNApolymerase, a plurality of nucleotides, a catalytic divalent cation, (inthe absence of added amplification primers), to generate double-strandedconcatemers, wherein the forming a flexing amplification reactionmixture is conducted with a temperature ramp-up, a flexing incubationtemperature, and a temperature ramp-down; (4) washing thedouble-stranded concatemer; and (5) repeating steps (1)-(4) at leastonce.

In some embodiments, the method for analyzing nucleic acids from asingle cell further comprise the step (g): sequencing at least a portionof the nucleic acid concatemer, including sequencing the target sequenceand the spatial barcode sequence, to determine the spatial location ofthe target nucleic acid in the single cell.

In some embodiments, the sequencing of step (g) comprises sequencing atleast a portion of the nucleic acid concatemers using an optical imagingsystem comprising a field-of-view (FOV) greater than 1.0 mm². In someembodiments, the sequencing of step (g) includes placing the single cellin a flow cell having walls (e.g., top or first wall, and bottom orsecond wall) and a gap in-between, where the gap can be filled with afluid, where the flow cell is positioned in a fluorescence opticalimaging system. The single cell has a thickness that may require usingthe imaging system to focus separately on the first and second surfacesof the flow cell, when using a traditional imaging system. For improvedimaging of the sequencing reaction of the nucleic acids from the singlecell, the flow cell can be positioned in a high performance fluorescenceimaging system, which comprises two or more tube lenses which aredesigned to provide optimal imaging performance for the first and secondsurfaces of the flow cell at two or more fluorescence wavelengths. Insome embodiments, the high-performance imaging system further comprisesa focusing mechanism configured to refocus the optical system betweenacquiring images of the first and second surfaces of the flow cell. Insome embodiments, the high performance imaging system is configured toimage two or more fields-of-view on at least one of the first flow cellsurface or the second flow cell surface.

In some embodiments, the sequencing of step (g) comprises: contactingthe plurality of nucleic acid concatemers with a plurality of sequencingprimers, a plurality of polymerases, and a plurality of multivalentmolecules, wherein each of the multivalent molecules comprise two ormore duplicates of a nucleotide moiety that are connected to a core viaa linker.

In some embodiments, the multivalent molecule comprises multiplenucleotides that are bound to a particle (or core) such as a polymer, abranched polymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

In some embodiments, the multivalent molecule comprises: (1) a core, and(2) a plurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms. In some embodiments, the spacer is attached to thelinker. In some embodiments, the linker is attached to the nucleotideunit. In some embodiments, the nucleotide unit comprises a base, sugarand at least one phosphate group, and wherein the linker is attached tothe nucleotide unit through the base. In some embodiments, the linkercomprises an aliphatic chain or an oligo ethylene glycol chain whereboth linker chains having 2-6 subunits and optionally the linkerincludes an aromatic moiety.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein the multiple nucleotide armshave the same type of nucleotide unit which is selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule further comprises aplurality of multivalent molecules which includes a mixture ofmultivalent molecules having two or more different types of nucleotidesselected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, and wherein individual nucleotide armscomprise a nucleotide unit having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position.

In some embodiments, the chain terminating moiety comprise an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety is selected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, the chain terminating moiety is cleavable/removablefrom the nucleotide unit.

In some embodiments, the chain terminating moiety is an azide, azido orazidomethyl group which are cleavable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP).

In some embodiments, the multivalent molecule comprises a core attachedto multiple nucleotide arms, wherein the core is labeled with detectablereporter moiety. In some embodiments, the detectable reporter moietycomprises a fluorophore.

In some embodiments, the core of the multivalent molecule comprises anavidin-like moiety and the core attachment moiety comprises biotin.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of nucleic acid concatemers with (i) aplurality of polymerases, (ii) at least one multivalent moleculecomprising two or more duplicates of a nucleotide moiety that areconnected to a core via a linker, and (iii) a plurality of sequencingprimers that hybridize with a portion of the concatemers, under acondition suitable for binding at least one polymerase and at least onesequencing primer to a portion of one of the nucleic acid concatemermolecules, and suitable for binding at least one of the nucleotidemoieties of the multivalent molecule to the 3′ end of the sequencingprimer at a position that is opposite a complementary nucleotide in theconcatemer molecule wherein the bound nucleotide moiety does notincorporate into the sequencing primer; (2) detecting and identifyingthe bound nucleotide moiety of the multivalent molecule therebydetermining the sequence of the concatemer molecule; (3) optionallyrepeating steps (1) and (2) at least once; (4) contacting the concatemermolecule with (i) a plurality of polymerases, and (ii) a plurality ofnucleotides, under a condition suitable binding at least one polymeraseto at least a portion of the concatemer molecule and suitable forbinding at least one of the nucleotides from the plurality to the 3′ends of the hybridized sequencing primers at a position that is oppositea complementary nucleotide in the concatemer molecule wherein the boundnucleotides incorporate into the hybridized sequencing primers; (5)optionally detecting the incorporated nucleotides; (6) optionallyidentifying the incorporation nucleotides thereby determining orconfirming the sequence of the concatemer; and (7) repeating steps(1)-(6) at least once.

In some embodiments, the sequencing of step (g) comprises: (1)contacting the plurality of immobilized concatemers with a plurality ofsequencing primers that hybridize with the sequencing primer bindingsequence, a plurality of polymerases, and a plurality of nucleotides,under a condition suitable for binding at least one polymerase and atleast one sequencing primer to a portion of the immobilized concatemer,and suitable for binding at least one of the nucleotides to the 3′ endof the sequencing primer at a position that is opposite a complementarynucleotide in the immobilized concatemer wherein the bound nucleotideincorporates into the 3′ end of the sequencing primer; (2) detecting andidentifying the incorporated nucleotide thereby determining the sequenceof the immobilized concatemer molecule; and (3) optionally repeatingsteps (1) and (2) at least once. In some embodiments, at least one ofthe nucleotides in the plurality of nucleotides comprises a chainterminating moiety at the sugar 2′ or 3′ position. In some embodiments,the chain terminating moiety is an azide, azido or azidomethyl groupwhich are cleavable with a phosphine compound. In some embodiments, thephosphine compound comprises a derivatized tri-alkyl phosphine moiety ora derivatized tri-aryl phosphine moiety. In some embodiments, thephosphine compound comprises Tris(2-carboxyethyl)phosphine (TCEP) orbis-sulfo triphenyl phosphine (BS-TPP).

In some embodiments, in any of the sequencing steps can be conducted byperforming a sequencing-by-binding procedure which comprises: (1)contacting a primed template nucleic acid (e.g., a primer hybridized toa nucleic acid concatemer) with a polymerase and a first combination oftwo or three types of test nucleotides under conditions that form astabilized ternary complex between the polymerase, primed templatenucleic acid and a test nucleotide that is complementary to the nextbase of the primed template nucleic acid; (2) detecting the ternarycomplex while precluding incorporation of test nucleotides into theprimer; (3) repeating steps (1) and (2) using the primed templatenucleic acid, a polymerase and a second combination of two or threetypes of test nucleotides, wherein the second combination is differentfrom the first combination; (4) incorporating into the primer, afterstep (c), a nucleotide that is complimentary to the next base; and (5)repeating steps (1) through (4) to identify/determine the sequence ofthe primed template nucleic acid.

In some embodiments, the first combination of two or three types of testnucleotides includes two, and only two, types of test nucleotides.Optionally, the second combination can also include two, and only two,types of test nucleotides.

In some embodiments, steps (1) and (2) are carried out serially for fourdifferent combinations of two types of test nucleotides, wherein eachdifferent nucleotide type is contacted with the primed template nucleicacid two times in aggregate. Alternatively, steps (1) and (2) can becarried out serially for six different combinations of two types of testnucleotides, wherein each different nucleotide type is present threetimes in aggregate.

Further provided is a method of determining the identity of the nextcorrect nucleotide for a primed template nucleic acid molecule (e.g., aprimer hybridized to a nucleic acid concatemer). The method includes thesteps of: (1) providing a template nucleic acid molecule primed with aprimer (e.g., a primer hybridized to a nucleic acid concatemer); (2)contacting the primed template nucleic acid molecule from step (1) witha first reaction mixture including a polymerase and at least one testnucleotide under conditions that (i) stabilize ternary complexesincluding the primed template nucleic acid molecule, the polymerase anda next correct nucleotide, while precluding incorporation of anynucleotide into the primer, and (ii) destabilize binary complexesincluding the primed template nucleic acid molecule and the polymerasebut not the next correct nucleotide; (3) detecting (e.g., monitoring)interaction of the polymerase with the primed template nucleic acidmolecule without chemical incorporation of any nucleotide into theprimer of the primed template nucleic acid molecule, to determinewhether a ternary complex formed in step (2); and (4) determiningwhether any of the test nucleotides is the next correct nucleotide forthe primed template nucleic acid molecule using the result of step (3).According to one generally preferred embodiment, the conditions thatstabilize ternary complexes while precluding incorporation of anynucleotide into the primer can be provided by including in the firstreaction mixture a non-catalytic metal ion that inhibits polymerization.

Single and multichannel fluorescence imaging modules and systems:Disclosed herein are single- and multichannel imaging systems thatprovide improved performance in terms of field-of-view, imageresolution, image quality across the field-of-view, dual-surfaceimaging, imaging duty cycle time, and imaging throughput for genomicsapplications such as nucleic acid sequencing. In some instances, theimaging modules or systems disclosed herein may comprise fluorescenceimaging modules or systems.

In some instances, the fluorescence imaging systems disclosed herein maycomprise a single fluorescence excitation light source (for providingexcitation light at a single wavelength or within a single excitationwavelength range) and an optical path configured to deliver theexcitation light to a sample (e.g., fluorescently-tagged nucleic acidmolecules or clusters thereof disposed on a substrate surface). In someinstances, the fluorescence imaging systems disclosed herein maycomprise a single fluorescence emission imaging and detection channel,e.g., an optical path configured to collect fluorescence emitted by thesample and deliver an image of the sample (e.g., an image of a substratesurface on which fluorescently-tagged nucleic acid molecules or clustersthereof are disposed) to an image sensor or other photodetection device.In some instances, the fluorescence imaging systems may comprise two,three, four, or more than four fluorescence excitation light sourcesand/or optical paths configured to deliver excitation light at two,three, four, or more than four excitation wavelengths (or within two,three, four, or more than four excitation wavelength ranges). In someinstances, the fluorescence imaging systems disclosed herein maycomprise two, three, four, or more than four fluorescence emissionimaging and detection channels configured to collect fluorescenceemitted by the sample at two, three, four, or more than four emissionwavelengths (or within two, three, four, or more than four emissionwavelength ranges and deliver an image of the sample (e.g., an image ofa substrate surface on which fluorescently-tagged nucleic acid moleculesor clusters thereof are disposed) to two, three, four, or more than fourimage sensors or other photodetection devices.

Dual surface imaging: In some instances, the imaging systems disclosedherein, including fluorescence imaging systems, may be configured toacquire high-resolution images of a single sample support structure orsubstrate surface. In some instances, the imaging systems disclosedherein, including fluorescence imaging systems, may be configured toacquire high-resolution images of two or more sample support structuresor substrate surfaces, e.g., two or more surfaces of a flow cell. Insome instances, the high-resolution images provided by the disclosedimaging systems may be used to monitor reactions occurring on the two ormore surfaces of the flow cell (e.g., nucleic acid hybridization,amplification, and/or sequencing reactions) as various reagents flowthrough the flow cell or around a flow cell substrate. FIG. 8A and FIG.8B provide schematic illustrations of such dual surface supportstructures. FIG. 8A shows a dual surface support structure such as aflow cell that includes an internal flow channel through which ananalyte or reagent can be flowed. The flow channel may be formed betweenfirst and second, top and bottom, and/or front and back layers such asfirst and second, top and bottom, and/or front and back plates as shown.One or more of the plates may include a glass plate, such as acoverslip, or the like. In some implementations, the layer comprisesborosilicate glass, quartz, or plastic. Interior surfaces of these topand bottom layers provide walls of the flow channel that assist inconfining the flow of analyte or reagent through the flow channel of theflow cell. In some designs, these interior surfaces are planar.Similarly, the top and bottom layers may be planar. In some designs, atleast one additional layer (not shown) is disposed between the top andbottom layers. This additional layer may have one or more pathways cuttherein that assist in defining one or more flow channels andcontrolling the flow of the analyte or reagent within the flow channel.Additional discussion of sample support structures, e.g., flow cells,can be found below.

FIG. 8A schematically illustrates a plurality of fluorescing samplesites on the first and second, top and bottom, and/or front and backinterior surfaces of the flow cell. In some implementations, reactionsmay occur at these at these sites to bind sample such that fluorescenceis emitted from these sites (note that FIG. 8A is schematic and notdrawn to scale; for example, the size and spacing of the fluorescingsample sites may be smaller than shown).

FIG. 8B shows another dual surface support structure having two surfacescontaining fluorescing sample sites to be imaged. The sample supportstructure comprises a substrate having first and second, top and bottom,and/or front and back exterior surfaces. In some designs, these exteriorsurfaces are planar. In various implementations, the analyte or reagentis flowed across these first and second exterior surfaces. FIG. 8Bschematically illustrates a plurality of fluorescing sample sites on thefirst and second, top and bottom, and/or front and back exteriorsurfaces of the sample support structure. In some implementations,reactions may occur at these at these sites to bind sample such thatfluorescence is emitted from these sites (note that FIG. 8B is schematicand not drawn to scale; for example, the size and spacing of thefluorescing sample sites may be smaller than shown).

In some instances, the fluorescence imaging modules and systemsdescribed herein may be configured to image such fluorescing samplesites on first and second surfaces at different distances from theobjective lens. In some designs, only one of the first or secondsurfaces is in focus at a time. Accordingly, in such designs, one of thesurfaces is imaged at a first time, and the other surface is imaged at asecond time. The focus of the fluorescence imaging module may be changedafter imaging one of the surfaces in order to image the other surfacewith comparable optical resolution, as the images of the two surfacesare not simultaneously in focus. In some designs, an opticalcompensation element may be introduced into the optical path between thesample support structure and the image sensor in order to image one ofthe two surfaces. The depth of field in such fluorescence imagingconfigurations may not be sufficiently large to include both the firstand second surfaces. In some implementations of the fluorescence imagingmodules described herein, both the first and second surfaces may beimaged at the same time, i.e., simultaneously. For example, thefluorescence imaging module may have a depth of field that issufficiently large to include both surfaces. In some instances, thisincreased depth of field may be provided by, for example, reducing thenumerical aperture of the objective lens (or microscope objective) aswill be discussed in more detail below.

As shown in FIGS. 8A and 8B, the imaging optics (e.g., an objectivelens) may be positioned at a suitable distance (e.g., a distancecorresponding to the working distance) from the first and secondsurfaces to form in-focus images of the first and second surfaces on animage sensor of a detection channel. As shown in the example of FIGS. 8Aand 8B, the first surface may be between said objective lens and thesecond surface. For example, as illustrated, the objective lens isdisposed above both the first and second surfaces, and the first surfaceis disposed above the second surface. The first and second surfaces, forexample, are at different depths. The first and second surfaces are atdifferent distances from any one or more of the fluorescence imagingmodule, the illumination and imaging module, imaging optics, or theobjective lens. The first and second surfaces are separated from eachother with the first surface spaced apart above the second surface. Inthe example shown, the first and second surfaces are planar surfaces andare separated from each other along a direction normal to said first andsecond planar surfaces. Also, in the example shown, said objective lenshas an optical axis and said first and second surfaces are separatedfrom each other along the direction of said optical axis. Similarly, theseparation between the first and second surfaces may correspond to thelongitudinal distance such as along the optical path of the excitationbeam and/or along an optical axis through the fluorescence imagingmodule and/or the objective lens. Accordingly, these two surfaces may beseparated by a distance from each other in the longitudinal (Z)direction, which may be along the direction of the central axis of theexcitation beam and/or the optical axis of the objective lens and/or thefluorescence imaging module. This separation may correspond, forexample, to a flow channel within a flow cell in some implementations.

In various designs, the objective lens (possibly in combination withanother optical component, e.g., a tube lens) have a depth of fieldand/or depth of focus that is at least as large as the longitudinalseparation (in the Z direction) between the first and second surfaces.The objective lens, alone or in combination with the additional opticalcomponent, may thus simultaneously form in-focus images of both thefirst and the second surface on an image sensor of one or more detectionchannels where these images have comparable optical resolution. In someimplementations, the imaging module may or may not need to be re-focusedto capture images of both the first and second surfaces with comparableoptical resolution. In some implementations, compensation optics neednot be moved into or out of an optical path of the imaging module toform in-focus images of the first and second surfaces. Similarly, insome implementations, one or more optical elements (e.g., lens elements)in the imaging module (e.g., the objective lens and/or a tube lens) neednot be moved, for example, in the longitudinal direction along the firstand/or second optical paths (e.g., along the optical axis of the imagingoptics) to form in-focus images of the first surface in comparison tothe location of said one or more optical element when used to formin-focus images of the second surface. In some implementations, however,the imaging module includes an autofocus system configured to provideboth the first and second surface in focus at the same time. In variousimplementations, the sample is in focus to sufficiently resolve thesample sites, which are closely spaced together in lateral directions(e.g., the X and Y directions). Accordingly, in various implementations,no optical element enters an optical path between the sample supportstructure (e.g., between a translation stage that supports the samplesupport structure) and an image sensor (or photodetector array) in theat least one detection channel in order to form in-focus images offluorescing sample sites on a first surface of the sample supportstructure and on a second surface of said sample support structure.Similarly, in various implementations, no optical compensation is usedto form an in-focus image of fluorescing sample sites on a first surfaceof the sample support structure on the image sensor or photodetectorarray that is not identical to optical compensation used to form anin-focus image of fluorescing sample sites on a second surface of thesample support structure on the image sensor or photodetector array.Additionally, in certain implementations, no optical element in anoptical path between the sample support structure (e.g., between atranslation stage that supports the sample support structure) and animage sensor in the at least one detection channel is adjusteddifferently to form an in-focus image of fluorescing sample sites on afirst surface of the sample support structure than to form an in-focusimage of fluorescing sample sites on a second surface of the samplesupport structure. Similarly, in some various implementations, nooptical element in an optical path between the sample support structure(e.g., between a translation stage that supports the sample supportstructure) and an image sensor in the at least one detection channel ismoved a different amount or a different direction to form an in-focusimage of fluorescing sample sites on the a first surface of the samplesupport structure on the image sensor than to form an in-focus image offluorescing sample sites on a second surface of said sample supportstructure on the image sensor. Any combination of the features ispossible. For example, in some implementations, in-focus images of theupper interior surface and the lower interior surface of the flow cellcan be obtained without moving an optical compensator into or out of anoptical path between the flow cell and the at least one image sensor andwithout moving one or more optical elements of the imaging system (e.g.,the objective and/or tube lens) along the optical path (e.g., opticalaxis) therebetween. For example, in-focus images of the upper interiorsurface and the lower interior surface of the flow cell can be obtainedwithout moving one or more optical elements of the tube lens into or outof the optical path, or without moving one or more optical elements ofthe tube lens along the optical path (e.g., optical axis) therebetween.

Any one or more of the fluorescence imaging module, the illuminationoptical path, the imaging optical path, the objective lens, or the tubelens may be designed to reduce or minimize optical aberration at twolocations such as two planes corresponding to two surfaces on a flowcell or other sample support structure, for example, where fluorescingsample sites are located. Any one or more of the fluorescence imagingmodule, the illumination optical path, the imaging optical path, theobjective lens, or the tube lens may be designed to reduce or minimizeoptical aberration at the selected locations or planes relative to otherlocations or planes, such as first and second surfaces containingfluorescing sample sites on a dual surface flow cell. For example, anyone or more of the fluorescence imaging module, the illumination opticalpath, the imaging optical path, the objective lens, or the tube lens maybe designed to reduce or minimize optical aberration at two depths orplanes located at different distances from the objective lens ascompared to the aberrations associated with other depths or planes atother distances from the objective lens. For example, optical aberrationmay be less for imaging the first and second surfaces than elsewhere ina region ranging from about 1 to about 10 mm from the objective lens.Additionally, any one or more of the fluorescence imaging module, theillumination optical path, the imaging optical path, the objective lens,or the tube lens may, in some instances, be configured to compensate foroptical aberration induced by transmission of emission light through oneor more portions of the sample support structure such as a layer thatincludes one of the surfaces on which sample adheres as well as possiblya solution that is in contact with the sample. This layer (e.g., acoverslip or the wall of a flow cell) may comprise, e.g., glass, quartz,plastic, or other transparent material having a refractive index andthat introduces optical aberration.

Accordingly, the imaging performance may be substantially the same whenimaging the first surface and second surface. For example, the opticaltransfer functions (OTF) and/or modulation transfer functions (MTF) maybe the substantially the same for imaging of the first and secondsurfaces. Either or both of these transfer functions may, for example,be within 20%, within 15%, within 10%, within 5%, within 2.5%, or within1% of each other, or within any range formed by any of these values atone or more specified spatial frequencies or when averaged over a rangeof spatial frequencies. Accordingly, an imaging performance metric maybe substantially the same for imaging the upper interior surface or thelower interior surface of the flow cell without moving an opticalcompensator into or out of an optical path between the flow cell and theat least one image sensor, and without moving one or more opticalelements of the imaging system (e.g., the objective and/or tube lens)along the optical path (e.g., optical axis) therebetween. For example,an imaging performance metric may be substantially the same for imagingthe upper interior surface or the lower interior surface of the flowcell without moving one or more optical elements of the tube lens intoor out of the optical path or without moving one or more opticalelements of the tube lens along the optical path (e.g., optical axis)therebetween. Additional discussion of MTF is included below and in U.S.Provisional Application No. 62/962,723 filed Jan. 17, 2020, which isincorporated herein by reference in its entirety.

It will be understood by those of skill in the art that the disclosedimaging modules or systems may, in some instances, be stand-aloneoptical systems designed for imaging a sample or substrate surface. Insome instances, they may comprise one or more processors or computers.In some instances, they may comprise one or more software packages thatprovide instrument control functionality and/or image processingfunctionality. In some instances, in addition to optical components suchas light sources (e.g., solid-state lasers, dye lasers, diode lasers,arc lamps, tungsten-halogen lamps, etc.), lenses, prisms, mirrors,dichroic reflectors, beam splitters, optical filters, optical bandpassfilters, light guides, optical fibers, apertures, and image sensors(e.g., complementary metal oxide semiconductor (CMOS) image sensors andcameras, charge-coupled device (CCD) image sensors and cameras, etc.),they may also include mechanical and/or optomechanical components, suchas X-Y translation stages, X-Y-Z translation stages, piezoelectricfocusing mechanisms, electro-optical phase plates, and the like. In someinstances, they may function as modules, components, sub-assemblies, orsub-systems of larger systems designed for, e.g., genomics applications(e.g., genetic testing and/or nucleic acid sequencing applications). Forexample, in some instances, they may function as modules, components,sub-assemblies, or sub-systems of larger systems that further compriselight-tight and/or other environmental control housings, temperaturecontrol modules, flow cells and cartridges, fluidics control modules,fluid dispensing robotics, cartridge- and/or microplate-handling(pick-and-place) robotics, one or more processors or computers, one ormore local and/or cloud-based software packages (e.g., instrument/systemcontrol software packages, image processing software packages, dataanalysis software packages), data storage modules, data communicationmodules (e.g., Bluetooth, WiFi, intranet, or internet communicationhardware and associated software), display modules, etc., or anycombination thereof. These additional components of larger systems,e.g., systems designed for genomics applications, will be discussed inmore detail below.

FIGS. 9A and 9B illustrate a non-limiting example of an illumination andimaging module 32 for multi-channel fluorescence imaging. Theillumination and imaging module 32 includes an objective lens 39, anillumination source 36, a plurality of detection channels 34, and afirst dichroic filter 38, which may comprise a dichroic reflector orbeam splitter. An autofocus system, which may include an autofocus laser33, for example, that projects a spot the size of which is monitored todetermine when the imaging system is in-focus may be included in somedesigns. Some or all components of the illumination and imaging module32 may be coupled to a baseplate 35.

The illumination or light source 36 may include any suitable lightsource configured to produce light of at least a desired excitationwavelength (discussed in more detail below). The light source may be abroadband source that emits light within one or more excitationwavelength ranges (or bands). The light source may be a narrowbandsource that emits light within one or more narrower wavelength ranges.In some instances, the light source may produce a single isolatedwavelength (or line) corresponding to the desired excitation wavelength,or multiple isolated wavelengths (or lines). In some instances, thelines may have some very narrow bandwidth. Example light sources thatmay be suitable for use in the illumination source 36 include, but arenot limited to, an incandescent filament, xenon arc lamp, mercury-vaporlamp, a light-emitting diode, a laser source such as a laser diode or asolid-state laser, or other types of light sources. As discussed below,in some designs, the light source may comprise a polarized light sourcesuch as a linearly polarized light source. In some implementations, theorientation of the light source is such that s-polarized light isincident on one or more surfaces of one or more optical components suchas the dichroic reflective surface of one or more dichroic filters.

The illumination source 36 may further include one or more additionaloptical components such as lenses, filters, optical fibers, or any othersuitable transmissive or reflective optics as appropriate to output anexcitation light beam having suitable characteristics toward a firstdichroic filter 38. For example, beam shaping optics may be included,for example, to receive light from a light emitter in the light sourceand produce a beam and/or provide a desired beam characteristic. Suchoptics may, for example, comprise a collimating lens configured toreduce the divergence of light and/or increase collimation and/or tocollimate the light.

In some implementations, multiple light sources are included in theillumination and imaging module 32. In some such implementations,different light sources may produce light having different spectralcharacteristics, for example, to excite different fluorescence dyes. Insome implementations, light produced by the different light sources maydirected to coincide and form an aggregate excitation light beam. Thiscomposite excitation light beam may be composed of excitation lightbeams from each of the light sources. The composite excitation lightbeam will have more optical power than the individual beams that overlapto form the composite beam. For example, in some implementations thatinclude two light sources that produce two excitation light beams, thecomposite excitation light beam formed from the two individualexcitation light beams may have optical power that is the sum of theoptical power of the individual beams. Similarly, in someimplementations, three, four, five or more light sources may beincluded, and these light sources may each output excitation light beamsthat together form a composite beam that that has an optical power thatis the sum of the optical power of the individual beams.

In some implementations, the light source 36 outputs a sufficientlylarge amount of light to produce sufficiently strong fluorescenceemission. Stronger fluorescence emission can increase thesignal-to-noise ratio (SNR) and the contrast-to-noise ratio (CNR) ofimages acquired by the fluorescence imaging module. In someimplementations, the output of the light source and/or an excitationlight beam derived therefrom (including a composite excitation lightbeam) may range in power from about 0.5 W to about 5.0 W, or more (aswill be discussed in more detail below).

Referring again to FIGS. 9A and 9B, the first dichroic filter 38 isdisposed with respect to the light source to receive light therefrom.The first dichroic filter may comprise a dichroic mirror, dichroicreflector, dichroic beam splitter, or dichroic beam combiner configuredto transmit light in a first spectral region (or wavelength range) andreflect light having a second spectral region (or wavelength range). Thefirst spectral region may include one or more spectral bands, e.g., oneor more spectral bands in the ultraviolet and blue wavelength ranges.Similarly, a second spectral region may include one or more spectralbands, e.g., one or more spectral bands extending from the green to redand infrared wavelengths. Other spectral regions or wavelength rangesare also possible.

In some implementations, the first dichroic filter may be configured totransmit light from the light source to a sample support structure suchas to a microscope slide, a capillary, a flow cell, a microfluidic chip,or other substrate or support structure. The sample support structuresupports and positions the sample, e.g., a composition comprising afluorescently-labeled nucleic acid molecule or complement thereof, withrespect to the illumination and imaging module 32. Accordingly, a firstoptical path extends from the light source to the sample via the firstdichroic filter. In various implementations, the sample supportstructure includes at least one surface on which the sample is disposedor to which the sample binds. In some instances, the sample may bedisposed within or bound to different localized regions or sites on theat least one surface of the sample support structure.

In some instances, the support structure may include two surfaceslocated at different distances from objective lens 39 (i.e., atdifferent positions or depths along the optical axis of objective lens39) on which the sample is disposed. As discussed below, for example, aflow cell may comprise a fluid channel formed at least in part by firstand second (e.g., upper and lower) interior surfaces, and the sample maybe disposed at localized sites on the first interior surface, the secondinterior surface, or both interior surfaces. The first and secondsurface may be separated by the region corresponding to the fluidchannel through which a solution flows, and thus be at differentdistances or depth with respect to objective lens 39 of the illuminationand imaging module 32.

The objective lens 39 may be included in the first optical path betweenthe first dichroic filter and the sample. This objective lens may beconfigured, for example, to have a focal length, working distance,and/or be positioned to focus light from the light source(s) onto thesample, e.g., onto a surface of the microscope slide, capillary, flowcell, microfluidic chip, or other substrate or support structure.Similarly, the objective lens 39 may be configured to have suitablefocal length, working distance, and/or be positioned to collect lightreflected, scattered, or emitted from the sample (e.g., fluorescenceemission) and to form an image of the sample (e.g., a fluorescenceimage).

In some implementations, objective lens 39 may comprise a microscopeobjective such as an off-the-shelf objective. In some implementations,objective lens 39 may comprise a custom objective. An example of acustom objective lens and/or custom objective-tube lens combination isdescribed below and in U.S. Provisional Application No. 62/962,723 filedon Jan. 17, 2020, which is incorporated herein by reference in itsentirety. The objective lens 39 may be designed to reduce or minimizeoptical aberration at two locations such as two planes corresponding totwo surfaces of a flow cell or other sample support structure. Theobjective lens 39 may be designed to reduce the optical aberration atthe selected locations or planes, e.g., the first and second surfaces ofa dual surface flow cell, relative to other locations or planes in theoptical path. For example, the objective lens 39 may be designed toreduce the optical aberration at two depths or planes located atdifferent distances from the objective lens as compared to the opticalaberrations associated with other depths or planes at other distancesfrom the objective. For example, in some instances, optical aberrationmay be less for imaging the first and second surfaces of a flow cellthan that exhibited elsewhere in a region spanning from 1 to 10 mm fromthe front surface of the objective lens. Additionally, a customobjective lens 39 may in some instances be configured to compensate foroptical aberration induced by transmission of fluorescence emissionlight through one or more portions of the sample support structure, suchas a layer that includes one or more of the flow cell surfaces on whicha sample is disposed, or a layer comprising a solution filling the fluidchannel of a flow cell. These layers may comprise, e.g., glass, quartz,plastic, or other transparent material having a refractive index, andwhich may introduce optical aberration.

In some implementations, objective lens 39 may have a numerical aperture(NA) of 0.6 or more (as discussed in more detail below). Such anumerical aperture may provide for reduced depth of focus and/or depthof field, improved background discrimination, and increased imagingresolution.

In some implementations, objective lens 39 may have a numerical aperture(NA) of 0.6 or less (as discussed in more detail below). Such anumerical aperture may provide for increased depth of focus and/or depthof field. Such increased depth of focus and/or depth of field mayincrease the ability to image planes separated by a distance such asthat that separates the first and second surfaces of a dual surface flowcell.

As discussed above, a flow cell may comprise, for example, first andsecond layers comprising first and second interior surfaces respectivelythat are separated by a fluid channel through which an analyte orreagent can flow. In some implementations, the objective lens 39 and/orillumination and imaging module 32 may be configured to provide a depthof field and/or depth of focus sufficiently large to image both thefirst and second interior surfaces of the flow cell, either sequentiallyby re-focusing the imaging module between imaging the first and secondsurfaces, or simultaneously by ensuring a sufficiently large depth offield and/or depth of focus, with comparable optical resolution. In someinstances, the depth of field and/or depth of focus may be at least aslarge or larger than the distance separating the first and secondsurfaces of the flow cell to be imaged, such as the first and secondinterior surfaces of the flow cell. In some instances, the first andsecond surfaces, e.g., the first and second interior surfaces of a dualsurface flow cell or other sample support structure, may be separated,for example, by a distance ranging from about 10 μm to about 700 μm, ormore (as will be discussed in more detail below). In some instances, thedepth of field and/or depth of focus may thus range from about 10 μm toabout 700 μm, or more (as will be discussed in more detail below).

In some designs, compensation optics (e.g., an “optical compensator” or“compensator”) may be moved into or out of an optical path in theimaging module, for example, an optical path by which light collected bythe objective lens 39 is delivered to an image sensor, to enable theimaging module to image the first and second surfaces of the dualsurface flow cell. The imaging module may be configured, for example, toimage the first surface when the compensation optics is included in theoptical path between the objective lens and an image sensor orphotodetector array configured to capture an image of the first surface.In such a design, the imaging module may be configured to image thesecond surface when the compensation optics is removed from or notincluded in the optical path between the objective lens 39 and the imagesensor or photodetector array configured to capture an image of thesecond surface. The need for an optical compensator may be morepronounced when using an objective lens 39 with a high numericalaperture (NA) value, e.g., for numerical aperture values of at least0.6, least 0.65, at least 0.7, at least 0.75, at least 0.8, at least0.85, at least 0.9, at least 0.95, at least 1.0, or higher. In someimplementations, the optical compensation optics (e.g., an opticalcompensator or compensator) comprises a refractive optical element suchas a lens, a plate of optically-transparent material such as glass, aplate of optically-transparent material such as glass, or in the case ofpolarized light beams, a quarter-wave plate or half-wave plate, etc.Other configurations may be employed to enable the first and secondsurfaces to be imaged at different times. For example, one or morelenses or optical elements may be configured to be translated in and outof, or along, an optical path between the objective lens 39 and theimage sensor.

In certain designs, however, the objective lens 39 is configured toprovide sufficiently large depth of focus and/or depth of field toenable the first and second surfaces to be imaged with comparableoptical resolution without such compensation optics moving into and outof an optical path in the imaging module, such as an optical pathbetween the objective lens and the image sensor or photodetector array.Similarly, in various designs, the objective lens 39 is configured toprovide sufficiently large depth of focus and/or depth of field toenable the first and second surfaces to be imaged with comparableoptical resolution without optics being moved, such as one or morelenses or other optical components being translated along an opticalpath in the imaging module, such as an optical path between theobjective lens and the image sensor or photodetector array. Examples ofsuch objective lenses will be described in more detail below.

In some implementations, the objective lens (or microscope objective) 39may be configured to have reduced magnification. The objective lens 39may be configured, for example, such that the fluorescence imagingmodule has a magnification of from less than 2× to less than 10× (aswill be discussed in more detail below). Such reduced magnification mayalter design constraints such that other design parameters can beachieved. For example, the objective lens 39 may also be configured suchthat the fluorescence imaging module has a large field-of-view (FOV)ranging, for example, from about 1.0 mm to about 5.0 mm (e.g., indiameter, width, length, or longest dimension) as will be discussed inmore detail below.

In some implementations, the objective lens 39 may be configured toprovide the fluorescence imaging module with a field-of-view asindicated above such that the FOV has diffraction-limited performance,e.g., less than 0.15 waves of aberration over at least 60%, 70%, 80%,90%, or 95% of the field, as will be discussed in more detail below.

In some implementations, the objective lens 39 may be configured toprovide the fluorescence imaging module with a field-of-view asindicated above such that the FOV has diffraction-limited performance,e.g., a Strehl ratio of greater than 0.8 over at least 60%, 70%, 80%,90%, or 95% of the field, as will be discussed in more detail below.

Referring again to FIGS. 9A and 9B, the first dichroic beam splitter orbeam combiner is disposed in the first optical path between the lightsource and the sample so as to illuminate the sample with one or moreexcitation beams. This first dichroic beam splitter or combiner is alsoin one or more second optical path(s) from the sample to the differentoptical channels used to detect the fluorescence emission. Accordingly,the first dichroic filter 38 couples the first optical path of theexcitation beam emitted by the illumination source 36 and second opticalpath of the emission light emitted by a sample specimen to the variousoptical channels where the light is directed to respective image sensorsor photodetector arrays for capturing images of the sample.

In various implementations, the first dichroic filter 38, e.g., firstdichroic reflector or beam splitter or beam combiner, has a passbandselected to transmit light from the illumination source 36 only within aspecified wavelength band or possibly a plurality of wavelength bandsthat include the desired excitation wavelength or wavelengths. Forexample, the first dichroic beam splitter 38 includes a reflectivesurface comprising a dichroic reflector that has spectral transmissivityresponse that is, e.g., configured to transmit light having at leastsome of the wavelengths output by the light source that form part of theexcitation beam. The spectral transmissivity response may be configurednot to transmit (e.g., instead to reflect) light of one or more otherwavelengths, for example, of one or more other fluorescence emissionwavelengths. In some implementations, the spectral transmissivityresponse may also be configured not to transmit (e.g., instead toreflect) light of one or more other wavelengths output by the lightsource. Accordingly, the first dichroic filter 38 may be utilized toselect which wavelength or wavelengths of light output by the lightsource reach the sample. Conversely, the dichroic reflector in the firstdichroic beam splitter 38 has a spectral reflectivity response thatreflects light having one or more wavelengths corresponding to thedesired fluorescence emission from the sample and possible reflectslight having one or more wavelengths output from the light source thatis not intended to reach the sample. Accordingly, in someimplementations, the dichroic reflector has a spectral transmissivitythat includes one or more pass bands to transmit the light to beincident on the sample and one or more stop bands that reflects lightoutside the pass bands, for example, light at one or more emissionwavelengths and possibly one or more wavelengths output by the lightsource that are not intended to reach the sample. Likewise, in someimplementations the dichroic reflector has a spectral reflectivity thatincludes one or more spectral regions configured to reflect one or moreemission wavelengths and possible one or more wavelengths output by thelight source that are not intended to reach the sample and includes oneor more regions that transmit light outside these reflection regions.The dichroic reflector included in the first dichroic filter 38 maycomprise a reflective filter such as an interference filter (e.g., aquarter-wave stack) configured to provide the appropriate spectraltransmission and reflection distributions. FIGS. 9A and 9B also show adichroic filter 38, which may comprise for example a dichroic beamsplitter or beam combiner, that may be used to direct the autofocuslaser 33 though the objective and to the sample support structure.

Although the imaging module 32 shown in FIGS. 9A and 9B and discussedabove is configured such that the excitation beam is transmitted by thefirst dichroic filter 38 to the objective lens 39, in some designs theillumination source 36 may be disposed with respect to the firstdichroic filter 38 and/or the first dichroic filter is configured (e.g.,oriented) such that the excitation beam is reflected by the firstdichroic filter 38 to the objective lens 39. Similarly, in some suchdesigns, the first dichroic filter 38 is configured to transmitfluorescence emission from the sample and possibly transmit light havingone or more wavelengths output from the light source that is notintended to reach the sample. As will be discussed below, a design wherethe fluorescence emission is transmitted instead of reflected maypotentially reduce wavefront error in the detected emission and/orpossibly have other advantages. In either case, in variousimplementations the first dichroic reflector 38 is disposed in thesecond optical path so as to receive fluorescence emission from thesample, at least some of which continues on to the detection channels34.

FIGS. 10A and 10B illustrate the optical paths within the multi-channelfluorescence imaging module of FIGS. 10A and 10B. In the example show inFIG. 10A and FIG. 10A, the detection channels 34 are disposed to receivefluorescence emission from a sample specimen that is transmitted by theobjective lens 39 and reflected by the first dichroic filter 38. Asreferred to above and described more below, in some designs thedetection channels 34 may be disposed to receive the portion of theemission light that is transmitted, rather than reflected, by the firstdichroic filter. In either case, the detection channels 34 may includeoptics for receiving at least a portion of the emission light. Forexample, the detection channels 34 may include one or more lenses, suchas tube lenses, and may include one or more image sensors or detectorssuch as photodetector arrays (e.g., CCD or CMOS sensor arrays) forimaging or otherwise producing a signal based on the received light. Thetube lenses may, for example, comprise one or more lens elementsconfigured to form an image of the sample onto the sensor orphotodetector array to capture an image thereof. Additional discussionof detection channels is included below and in U.S. ProvisionalApplication No. 62/962,723, filed Jan. 17, 2020, which is incorporatedherein by reference in its entirety. In some instances, improved opticalresolution may be achieved using an image sensor having relatively highsensitivity, small pixels, and high pixel count, in conjunction with asuitable sampling scheme, which may include oversampling orundersampling.

FIGS. 10A and 10B are ray tracing diagrams illustrating optical paths ofthe illumination and imaging module 32 of FIGS. 9A and 9B. FIG. 10Acorresponds to a top view of the illumination and imaging module 32.FIG. 10B corresponds to a side view of the illumination and imagingmodule 32. The illumination and imaging module 32 illustrated in thesefigures includes four detection channels 34. However, it will beunderstood that the disclosed illumination and imaging modules mayequally be implemented in systems including more or fewer than fourdetection channels 34. For example, the multi-channel systems disclosedherein may be implemented with as few as one detection channel 34, or asmany as two detection channels 34, three detection channels 34, fourdetection channels 34, five detection channels 34, six detectionchannels 34, seven detection channels 34, eight detection channels 34,or more than eight detection channels 34, without departing from thespirit or scope of the present disclosure.

The non-limiting example of imaging module 32 illustrated in FIGS. 10Aand 10B includes four detection channels 34, a first dichroic filter 38that reflects a beam 45 of emission light, a second dichroic filter(e.g., a dichroic beam splitter) 44 that splits the beam 45 into atransmitted portion and a reflected portion, and two channel-specificdichroic filters (e.g., dichroic beam splitters) 43 that further splitthe transmitted and reflected portions of the beam 45 among individualdetection channels 34. The dichroic reflecting surface in the dichroicbeam splitters 44 and 43 for splitting the beam 45 among detectionchannels are shown disposed at 45 degrees relative to a central beamaxis of the beam 45 or an optical axis of the imaging module. However,as discussed below, an angle smaller than 45 degrees may be employed andmay offer advantages such as sharper transitions from pass band to stopband.

The different detection channels 34 includes imaging devices 41, whichmay include an image sensor or photodetector array (e.g., a CCD or CMOSdetector array). The different detection channels 34 further includeoptics 42 such as lenses (e.g., one or more tube lenses each comprisingone or more lens elements) disposed to focus the portion of the emissionlight entering the detection channel 34 at a focal plane coincident witha plane of the photodetector array 41. The optics 42 (e.g., a tube lens)combined with the objective lens 39 are configured to form an image ofthe sample onto the photodetector array 41 to capture an image of thesample, for example, an image of a surface on the flow cell or othersample support structure after the sample has bound to that surface.Accordingly, such an image of the sample may comprise a plurality offluorescent emitting spots or regions across a spatial extent of thesample support structure where the sample is emitting fluorescencelight. The objective lens 39 together with the optics 42 (e.g., tubelens) may provide a field of view (FOV) that includes a portion of thesample or the entire sample. Similarly, the photodetector array 41 ofthe different detection channels 34 may be configured to capture imagesof a full field of view (FOV) provided by the objective lens and thetube lens, or a portion thereof. In some implementations, thephotodetector array 41 of some or all detection channels 34 can detectthe emission light emitted by a sample disposed on the sample supportstructure, e.g., a surface of the flow cell, or a portion thereof andrecord electronic data representing an image thereof. In someimplementations, the photodetector array 41 of some or all detectionchannels 34 can detect features in the emission light emitted by aspecimen without capturing and/or storing an image of the sampledisposed on the flow cell surface and/or of the full field of view (FOV)provided by the objective lens and optics 42 and/or 40 (e.g., elementsof a tube lens). In some implementations, the FOV of the disclosedimaging modules (e.g., that provided by the combination of objectivelens 39 and optics 42 and/or 40) may range, for example, between about 1mm and 5 mm (e.g., in diameter, width, length, or longest dimension) aswill be discussed below. The FOV may be selected, for example, toprovide a balance between magnification and resolution of the imagingmodule and/or based on one or more characteristics of the image sensorsand/or objective lenses. For example, a relatively smaller FOV may beprovided in conjunction with a smaller and faster imaging sensor toachieve high throughput.

Referring again to FIGS. 10A and 10B, in some implementations, theoptics 42 in the detection channel (e.g., the tube lens) may beconfigured to reduce optical aberration in images acquired using optics42 in combination with objective lens 39. In some implementationscomprising multiple detection channels for imaging at different emissionwavelengths, the optics 42 (e.g., the tube lens) for different detectionchannels have different designs to reduce aberration for the respectiveemission wavelengths at which that particular channel is configured toimage. In some implementations, the optics 42 (e.g., the tube lens) maybe configured to reduce aberrations when imaging a specific surface(e.g., a plane, object plane, etc.) on the sample support structurecomprising fluorescing sample sites disposed thereon as compared toother locations (e.g., other planes in object space). Similarly, in someimplementations, the optics 42 (e.g., the tube lens) may be configuredto reduce aberrations when imaging first and second surfaces (e.g.,first and second planes, first and second object planes, etc.) on a dualsurface sample support structure (e.g., a dual surface flow cell) havingfluorescing sample sites disposed thereon as compared to other locations(e.g., other planes in object space). For example, the optics 42 in thedetection channel (e.g., tube lens) may be designed to reduce theaberration at two depths or planes located at different distances fromthe objective lens as compared to the aberrations associated with otherdepths or planes at other distances from the objective. For example,optical aberration may be less for imaging the first and second surfacesthan elsewhere in a region from about 1 to about 10 mm from theobjective lens. Additionally, custom optic 42 in the detection channel(e.g., a tube lens) may in some embodiments be configured to compensatefor aberration induced by transmission of emission light through one ormore portions of the sample support structure such as a layer thatincludes one of the surfaces on which the sample is disposed as well aspossibly a solution adjacent to and in contact with the surface on whichthe sample is disposed. The layer comprising one of the surfaces onwhich the sample is disposed may comprise, e.g., glass, quartz, plastic,or other transparent material having a refractive index, and whichintroduces optical aberration. Custom optic 42 in the detection channel(e.g., the tube lens), for example, may in some implementations beconfigured to compensate for optical aberration induced by a samplesupport structure, e.g., a coverslip or flow cell wall, or other samplesupport structure components, as well as possibly a solution adjacent toand in contact with the surface on which the sample is disposed.

In some implementations, the optics 42 in the detection channel (e.g., atube lens) are configured to have reduced magnification. The optics 42in the detection channel (e.g., a tube lens) may be configured, forexample, such that the fluorescence imaging module has a magnificationof less than, for example, 10×, as will be discussed further below. Suchreduced magnification may alter design constraints such that otherdesign parameters can be achieved. For example, the optics 42 (e.g., atube lens) may also be configured such that the fluorescence imagingmodule has a large field-of-view (FOV), for example, of at least 1.0 mmor larger (e.g., in diameter, width, length, or longest dimension), aswill be discussed further below.

In some implementations, the optics 42 (e.g., a tube lens) may beconfigured to provide the fluorescence imaging module with afield-of-view as indicated above such that the FOV has less than 0.15waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of thefield, as will be discussed further below.

Referring again to FIGS. 10A and 10B, in various implementations, asample is located at or near a focal position 46 of the objective lens39. As described above with reference to FIGS. 9A and 9B, a light sourcesuch as a laser source provides an excitation beam to the sample toinduce fluorescence. At least a portion of fluorescence emission iscollected by the objective lens 39 as emission light. The objective lens39 transmits the emission light toward the first dichroic filter 38,which reflects some or all of the emission light as the beam 45 incidentupon the second dichroic filter 44 and to the different detectionchannels, each comprising optics 42 that form an image of the sample(e.g., a plurality of fluorescing sample sites on a surface of a samplesupport structure) onto a photodetector array 41.

As discussed above, in some implementations, the sample supportstructure comprises a flow cell such as a dual surface flow cell havingtwo surfaces (e.g., two interior surfaces, a first surface and a secondsurface, etc.) containing sample sites that emit fluorescent emission.These two surfaces may be separated by a distance from each other in thelongitudinal (Z) direction along the direction of the central axis ofthe excitation beam and/or the optical axis of the objective lens. Thisseparation may correspond, for example, to a flow channel within theflow cell. Analytes or reagents may be flowed through the flow channeland contact the first and second interior surfaces of the flow cell,which may thereby be contacted with a binding composition such thatfluorescence emission is radiated from a plurality of sites on the firstand second interior surfaces. The imaging optics (e.g., objective lens39) may be positioned at a suitable distance (e.g., a distancecorresponding to the working distance) from the sample to form in-focusimages of the sample on one or more detector arrays 41. As discussedabove, in various designs, the objective lens 39 (possibly incombination with the optics 42) may have a depth of field and/or depthof focus that is at least as large as the longitudinal separationbetween the first and second surfaces. The objective lens 39 and theoptics 42 (of each detection channel) can thus simultaneously formimages of both the first and the second flow cell surfaces on thephotodetector array 41, and these images of the first and secondsurfaces are both in focus and have comparable optical resolution (ormay be brought into focus with only minor refocusing of the objects toacquire images of the first and second surfaces that have comparableoptical resolution). In various implementations, compensation opticsneed not be moved into or out of an optical path of the imaging module(e.g., into or out of the first and/or second optical paths) to formin-focus images of the first and second surfaces that are of comparableoptical resolution. Similarly, in various implementations, one or moreoptical elements (e.g., lens elements) in the imaging module (e.g., theobjective lens 39 or optics 42) need not be moved, for example, in thelongitudinal direction along the first and/or second optical paths toform in-focus images of the first surface in comparison to the locationof said one or more optical elements when used to form in-focus imagesof the second surface. In some implementations, the imaging moduleincludes an autofocus system configured to quickly and sequentiallyrefocus the imaging module on the first and/or second surface such thatthe images have comparable optical resolution. In some implementations,objective lens 39 and/or optics 42 are configured such that both thefirst and second flow cell surfaces are in focus simultaneously withcomparable optical resolution without moving an optical compensator intoor out of the first and/or second optical path, and without moving oneor more lens elements (e.g., objective lens 39 and/or optics 42 (such asa tube lens) longitudinally along the first and/or second optics path.In some implementations, images of the first and/or second surfaces,acquired either sequentially (e.g., with refocusing between surfaces) orsimultaneously (e.g., without refocusing between surfaces) using thenovel objective lens and/or tube lens designs disclosed herein, may befurther processed using a suitable image processing algorithm to enhancethe effective optical resolution of the images such that the images ofthe first and second surfaces have comparable optical resolution. Invarious implementations, the sample plane is sufficiently in focus toresolve sample sites on the first and/or second flow cell surfaces, thesample sites being closely spaced in lateral directions (e.g., in the Xand Y directions).

As discussed above, the dichroic filters may comprise interferencefilters that selectively transmit and reflect light of differentwavelengths based on the principle of thin-film interference, usinglayers of optical coatings having different refractive indices andparticular thickness. Accordingly, the spectral response (e.g.,transmission and/or reflection spectra) of the dichroic filtersimplemented within multi-channel fluorescence imaging modules may be atleast partially dependent upon the angle of incidence, or range ofangles of incidence (e.g., dependent on beam diameter and/or beamdivergence), at which the light of the excitation and/or emission beamsare incident upon the dichroic filters. Such effects may be especiallysignificant with respect to the dichroic filters of the detectionoptical path (e.g., the dichroic filters 44 and 43 of FIGS. 10A and10B).

In some implementations, the focal length of the objective lens that issuitable for producing a narrow beam diameter with minimal divergencethat results in sharper may be longer than those typically employed influorescence microscopes or imaging systems. For example, in someimplementations, the focal length of the objective lens may rangebetween 20 mm and 40 mm, as will be discussed further below. In oneexample, an objective lens 39 having a focal length of 36 mm may producea beam 45 characterized by a divergence small enough that light acrossthe full diameter of the beam 45 is incident upon the second dichroicfilter 38 at angles within 2.5 degrees of the angle of incidence of thecentral beam axis.

In some implementations of the disclosed imaging modules, thepolarization state of the excitation beam may be utilized to furtherimprove the performance of the multi-channel fluorescence imagingmodules disclosed herein. Referring again to FIGS. 9A and 9B, forexample, some implementations of the multi-channel fluorescence imagingmodules disclosed herein have an epifluorescence configuration in whicha first dichroic filter 38 merges the optical paths of the excitationbeam and the beam of emission light such that both the excitation andemission light are transmitted through the objective lens 39. Asdiscussed above, the illumination source 36 may include a light sourcesuch as a laser or other source which provides the light that forms theexcitation beam. In some designs, the light source comprises a linearlypolarized light source and the excitation beam may be linearlypolarized. In some designs, polarization optics are included to polarizethe light and/or rotate the polarization of the light. For example, apolarizer such as a linear polarizer may be included in an optical pathof the excitation beam to polarize the excitation beam. Retarders suchas half wave retarders or a plurality of quarter wave retarders orretarders having other amounts of retardance may be included to rotatethe linear polarization in some designs.

The linearly polarized excitation beam, when it is incident upon anydichroic filter or other planar interface, may be p-polarized (e.g.,having an electric field component parallel to the plane of incidence),s-polarized (e.g., having an electric field component normal to theplane of incidence), or may have a combination of p-polarization ands-polarization states within the beam. The p- or s-polarization state ofthe excitation beam may be selected and/or changed by selecting theorientation of the illumination source 36 and/or one or more componentsthereof with respect to the first dichroic filter 38 and/or with respectto any other surfaces with which the excitation beam will interact. Insome implementations where the light source output linearly polarizedlight, the light source can be configured to provide s-polarized light.For example, the light source may comprise an emitter such as asolid-state laser or a laser diode that may be rotated about its opticalaxis or the central axis of the beam to orient the linearly polarizedlight output therefrom. Alternatively, or in addition, retarders may beemployed to rotate the linear polarization about the optical axis or thecentral axis of the beam. As discussed above, in some implementations,for example when the light source does not output polarized light, apolarizer disposed in the optical path of the excitation beam canpolarize the excitation beam. In some designs, for example, a linearpolarizer is disposed in the optical path of the excitation beam. Thispolarizer may be rotated to provide the proper orientation of the linearpolarization to provide s-polarized light.

In some designs, the linear polarization is rotated about the opticalaxis or the central axis of the beam such that s-polarization isincident on the dichroic reflector of the dichroic beam splitter. Whens-polarized light is incident on the dichroic reflector of the dichroicbeam splitter the transition between the pass band and the stop band issharper as opposed to when p-polarized light is incident on the dichroicreflector of the dichroic beam splitter.

As discussed above, in some implementation, a polarizer such as a linearpolarizer may be used to polarize the excitation beam. This polarizermay be rotated to provide an orientation of the linearly polarized lightcorresponding to s-polarized light. Also as discussed above, in someimplementations, other approaches to rotating the linearly polarizedlight may be used. For example, optical retarders such as half waveretarders or multiple quarter wave retarders may be used to rotate thepolarization direction. Other arrangements are also possible.

As discussed elsewhere herein, reducing the numerical aperture (NA) ofthe fluorescence imaging module and/or of the objective lens mayincrease the depth of field to enable the comparable imaging of the twosurfaces. FIGS. 11A-B, FIGS. 11A-B, and FIGS. 13A-B show how the MTF ismore similar at first and second surfaces separated by 1 mm of glass forlower numerical apertures than for larger numerical apertures. FIGS. 11Aand 11B show the MTF at first (FIG. 11A) and second (FIG. 11B) surfacesfor an NA of 0.3. FIGS. 12A and 12B show the MTF at first (FIG. 12A) andsecond (FIG. 12B) surfaces for an NA of 0.5. FIGS. 13A and 13B show theMTF at first (FIG. 13A) and second (FIG. 13B) surfaces for an NA of 0.7.The first and second surfaces in each of these figures correspond to,e.g., the top and bottom surfaces of a flow cell.

FIGS. 14A-B provide plots of the calculated Strehl ratio (i.e., theratio of peak light intensity focused or collected by the optical systemversus that focused or collected by an ideal optical system and pointlight source) for imaging a second flow cell surface through a firstflow cell surface. FIG. 14A shows a plot of the Strehl ratios forimaging a second flow cell surface through a first flow cell surface asa function of the thickness of the intervening fluid layer (fluidchannel height) for different objective lens and/or optical systemnumerical apertures. As shown, the Strehl ratio decreases withincreasing separation between the first and second surfaces. One of thesurfaces would thus have deteriorated image quality with increasingseparation between the two surfaces. The decrease in second surfaceimaging performance with increased separation distance between the twosurfaces is reduced for imaging systems having smaller numeral aperturesas compared to those having larger numerical apertures. FIG. 14B shows aplot of the Strehl ratio as a function of numerical aperture for imaginga second flow cell surface through a first flow cell surface and anintervening layer of water having a thickness of 0.1 mm. The loss ofimaging performance at higher numerical apertures may be attributed tothe increased optical aberration induced by the fluid for the secondsurface imaging. With increasing NA, the increased optical aberrationintroduced by the fluid for the second surface imaging degrades theimage quality significantly. In general, however, reducing the numeralaperture of the optical system reduces the achievable resolution. Thisloss of image quality can be at least partially offset by providing anincreased sample plane (or object plane) contrast-to-noise ratio, forexample, by using chemistries for nucleic acid sequencing applicationsthat enhance the fluorescence emission for labeled nucleic acid clustersand/or that reduce background fluorescence emission. In some instances,for example, sample support structures comprising hydrophilic substratematerials and/or hydrophilic coatings may be employed. In some cases,such hydrophilic substrates and/or hydrophilic coatings may reducebackground noise. Additional discussion of sample support structures,hydrophilic surfaces and coatings, and methods for enhancingcontrast-to-noise ratios, e.g., for nucleic acid sequencingapplications, can be found below.

In some implementations, any one or more of the fluorescence imagingsystem, the illumination and imaging module 32, the imaging optics(e.g., optics 42), the objective lens, and/or the tube lens isconfigured to have reduced magnification, such as a magnification ofless than 10×, as will be discussed further below. Such reducedmagnification may adjust design constraints such that other designparameters can be achieved. For example, any one or more of thefluorescence microscope, illumination and imaging module 32, the imagingoptics (e.g., optics 42), the objective lens or the tube lens may alsobe configured such that the fluorescence imaging module has a largefield-of-view (FOV), for example, a field-of-view of at least 3.0 mm orlarger (e.g., in diameter, width, height, or longest dimension), as willbe discussed further below. Any one or more of the fluorescence imagingsystem, the illumination and imaging module 32, the imaging optics(e.g., optics 42), the objective lens and/or the tube lens may beconfigured to provide the fluorescence microscope with such afield-of-view such that the FOV has less than, e.g., 0.1 waves ofaberration over at least 80% of field. Similarly, any one or more of thefluorescence imaging system, illumination and imaging module 32, theimaging optics (e.g., optics 42), the objective lens and/or the tubelens may be configured such that the fluorescence imaging module hassuch a FOV and is diffraction limited or is diffraction limited oversuch an FOV.

As discussed above, in various implementations, a large field-of-view(FOV) is provided by the disclosed optical systems. In someimplementations, obtaining an increased FOV is facilitated in part bythe use of larger image sensors or photodetector arrays. Thephotodetector array, for example, may have an active area with adiagonal of at least 15 mm or larger, as will be discussed furtherbelow. As discussed above, in some implementations the disclosed opticalimaging systems provide a reduced magnification, for example, of lessthan 10× which may facilitate large FOV designs. Despite the reducedmagnification, the optical resolution of the imaging module may still besufficient as detector arrays having small pixel size or pitch may beused. The pixel size and/or pitch may, for example, be about 5 μm orless, as will be discussed in more detail below. In someimplementations, the pixel size is smaller than twice the opticalresolution provided by the optical imaging system (e.g., objective andtube lens) to satisfy the Nyquist theorem. Accordingly, the pixeldimension and/or pitch for the image sensor(s) may be such that aspatial sampling frequency for the imaging module is at least twice anoptical resolution of the imaging module. For example, the spatialsampling frequency for the photodetector array may be is at least 2times, at least 2.5 times, at least 3 times, at least 4 times, or atleast 5 times the optical resolution of the fluorescence imaging module(e.g., the illumination and imaging module, the objective and tube lens,the object lens and optics 42 in the detection channel, the imagingoptics between the sample support structure or stage configured tosupport the sample support stage and the photodetector array) or anyspatial sampling frequency in a range between any of these values.

Although a wide range of features are discussed herein with respect tofluorescence imaging modules, any of the features and designs describeherein may be applied to other types of optical imaging systemsincluding without limitation bright-field and dark-field imaging and mayapply to luminescence or phosphorescence imaging.

Improved or optimized objective and/or tube lens for use with thickercoverslips: Existing design practice includes the design of objectivelenses and/or use of commonly available off-the-shelf microscopeobjectives to optimize image quality when images are acquired throughthin (e.g., <200 μm thick) microscope coverslips. When used to image onboth sides of a fluidic channel or flow cell, the extra height of thegap between the two surfaces (i.e., the height of the fluid channel;typically, about 50 μm to 200 μm) introduces optical aberration inimages captured for the non-optimal side of the fluidic channel, therebycausing lower optical resolution. This is primarily because theadditional gap height is significant compared to the optimal coverslipthickness (typical fluid channel or gap heights of 50-200 μm vs.coverslip thicknesses of <200 μm). Another common design practice is toutilize an additional “compensator” lens in the optical path whenimaging is to be performed on the non-optimal side of the fluid channelor flow cell. This “compensator” lens and the mechanism required to moveit in or out of the optical path so that either side of the flow cellmay be imaged further increases system complexity and imaging systemdown time, and potentially degrades image quality due to vibration, etc.

In the present disclosure, the imaging system is designed forcompatibility with flow cell consumables that comprise a thickercoverslip or flow cell wall (thickness≥700 μm). The objective lensdesign may be improved or optimized for a coverslip that is equal to thetrue cover slip thickness plus half of the effective gap thickness(e.g., 700 μm+½*fluid channel (gap) height). This design significantlyreduces the effect of gap height on image quality for the two surfacesof the fluid channel and balances the optical quality for images of thetwo surfaces, as the gap height is small relative to the total coverslipthickness and thus its impact on optical quality is reduced.

Additional advantages of using a thicker coverslip include improvedcontrol of thickness tolerance error during manufacturing, and a reducedlikelihood that the coverslip undergoes deformation due to thermal andmounting-induced stress. Coverslip thickness error and deformationadversely impact imaging quality for both the top surface and the bottomsurface of a flow cell.

To further improve the dual surface imaging quality for sequencingapplications, our optical system design places a strong emphasis onimproving or optimizing MTF (e.g., through improving or optimizing theobjective lens and/or tube lens design) in the mid- to high-spatialfrequency range that is most suitable for imaging and resolving smallspots or clusters.

Improved or optimized tube lens design for use in combination withcommercially-available, off-the-shelf objectives: For low-cost sequencerdesign, the use of a commercially-available, off-the-shelf objectivelens may be preferred due to its relatively low price. However, as notedabove, low-cost, off-the-shelf objectives are mostly optimized for usewith thin coverslips of about 170 μm in thickness. In some instances,the disclosed optical systems may utilize a tube lens design thatcompensates for a thicker flow cell coverslip while enabling high imagequality for both interior surfaces of a flow cell in dual-surfaceimaging applications. In some instances, the tube lens designs disclosedherein enable high quality imaging for both interior surfaces of a flowcell without moving an optical compensator into or out of the opticalpath between the flow cell and an image sensor, without moving one ormore optical elements or components of the tube lens along the opticalpath, and without moving one or more optical elements or components ofthe tube lens into or out of the optical path.

FIG. 15 provides an optical ray tracing diagram for a low lightobjective lens design that has been improved or optimized for imaging asurface on the opposite side of a 0.17 mm thick coverslip. The plot ofmodulation transfer function for this objective, shown in FIG. 16,indicates near-diffraction limited imaging performance when used withthe designed—for 0.17 mm thick coverslip.

FIG. 17 provides a plot of the modulation transfer function for the sameobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.3 mm thickcoverslip. The relatively minor deviations of MTF value over the spatialfrequency range of about 100 to about 800 lines/mm (or cycles/mm)indicates that the image quality obtained even when using a 0.3 mm thickcoverslip is still reasonable.

FIG. 18 provides a plot of the modulation transfer function for the sameobjective lens illustrated in FIG. 15 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid (i.e., under the kind of conditions encountered for dual-sideimaging of a flow cell when imaging the far surface). As can be seen inthe plot of FIG. 18, imaging performance is degraded, as indicated bythe deviations of the MTF curves from those for the an ideal,diffraction-limited case over the spatial frequency range of about 50lp/mm to about 900 lp/mm.

FIG. 19 and FIG. 20 provide plots of the modulation transfer function asa function of spatial frequency for the upper (or near) interior surface(FIG. 19) and lower (or far) interior surface (FIG. 20) of a flow cellwhen imaged using the objective lens illustrated in FIG. 15 through a1.0 mm thick coverslip, and when the upper and lower interior surfacesare separated by a 0.1 mm thick layer of aqueous fluid. As can be seen,imaging performance is significantly degraded for both surfaces.

FIG. 21 provides a ray tracing diagram for a tube lens design which, ifused in conjunction with the objective lens illustrated in FIG. 15,provides for improved dual-side imaging through a 1 mm thick coverslip.The optical design 47 comprising a compound objective (lens elements 49,50, 51, 52, 53, 54, 55, 56, and 57) and a tube lens (lens elements 58,59, 60, and 61) is improved or optimized for use with flow cellscomprising a thick coverslip (or wall), e.g., greater than 700 μm thick,and a fluid channel thickness of at least 50 μm, and transfers the imageof an interior surface from the flow cell 48 to the image sensor 62 withdramatically improved optical image quality and higher CNR.

In some instances, the tube lens (or tube lens assembly) may comprise atleast two optical lens elements, at least three optical lens elements,at least four optical lens elements, at least five optical lenselements, at least six optical lens elements, at least seven opticallens elements, at least eight optical lens elements, at least nineoptical lens elements, at least ten optical lens elements, or more,where the number of optical lens elements, the surface geometry of eachelement, and the order in which they are placed in the assembly isimproved or optimized to correct for optical aberrations induced by thethick wall of the flow cell, and in some instances, allows one to use acommercially-available, off-the-shelf objective while still maintaininghigh-quality, dual-side imaging capability.

In some instances, as illustrated in FIG. 21, the tube lens assembly maycomprise, in order, a first asymmetric convex-convex lens 58, a secondconvex-plano lens 59, a third asymmetric concave-concave lens 60, and afourth asymmetric convex-concave lens 61.

FIG. 22 and FIG. 23 provide plots of the modulation transfer function asa function of spatial frequency for the upper (or near) interior surface(FIG. 22) and lower (or far) interior surface (FIG. 23) of a flow cellwhen imaged using the objective lens (corrected for a 0.17 mm coverslip)and tube lens combination illustrated in FIG. 21 through a 1.0 mm thickcoverslip, and when the upper and lower interior surfaces are separatedby a 0.1 mm thick layer of aqueous fluid. As can be seen, the imagingperformance achieved is nearly that expected for a diffraction-limitedoptical design.

Imaging channel-specific tube lens adaptation or optimization: Inimaging system design, it is possible to improve or optimize both theobjective lens and the tube lens in the same wavelength region for allimaging channels. Typically, the same objective lens is shared by allimaging channels, and each imaging channel either uses the same tubelens or has a tube lens that shares the same design.

In some instances, the imaging systems disclosed herein may furthercomprise a tube lens for each imaging channel where the tube lens hasbeen independently improved or optimized for the specific imagingchannel to improve image quality, e.g., to reduce or minimize distortionand field curvature, and improve depth-of-field (DOF) performance foreach channel. Because the wavelength range (or bandpass) for eachspecific imaging channel is much narrower than the combined wavelengthrange for all channels, the wavelength- or channel-specific adaptationor optimization of the tube lens used in the disclosed systems resultsin significant improvements in imaging quality and performance. Thischannel-specific adaptation or optimization results in improved imagequality for both the top and bottom surfaces of the flow cell indual-side imaging applications.

Dual-side imaging w/o fluid present in flow cell: For optimal imagingperformance of both top and bottom interior surfaces of a flow cell, amotion-actuated compensator is typically required to correct for opticalaberrations induced by the fluid in the flow cell (typically comprisinga fluid layer thickness of about 50-200 μm). In some instances of thedisclosed optical system designs, the top interior surface of the flowcell may be imaged with fluid present in the flow cell. Once thesequencing chemistry cycle has been completed, the fluid may beextracted from the flow cell for imaging of the bottom interior surface.Thus, in some instances, even without the use of a compensator, theimage quality for the bottom surface is maintained.

Compensation for optical aberration and/or vibration usingelectro-optical phase plates: In some instances, dual-surface imagequality may be improved without requiring the removal of the fluid fromthe flow cell by using an electro-optical phase plate (or othercorrective lens) in combination with the objective to cancel the opticalaberrations induced by the presence of the fluid. In some instances, theuse of an electro-optical phase plate (or lens) may be used to removethe effects of vibration arising from the mechanical motion of amotion-actuated compensator and may provide faster image acquisitiontimes and sequencing cycle times for genomic sequencing applications.

Improved contrast-to-noise ratio (CNR), field-of-view (FOV), spectralseparation, and timing design to increase or maximize informationtransfer and throughput: Another way to increase or maximize informationtransfer in imaging systems designed for genomics applications is toincrease the size of the field-of-view (FOV) and reduce the timerequired to image a specific FOV. With typical large NA optical imagingsystems, it may be common to acquire images for fields-of-view that areon the order of 1 mm² in area, where in the presently disclosed imagingsystem designs large FOV objectives with long working distances arespecified to enable imaging of areas of 2 mm² or larger.

In some cases, the disclosed imaging systems are designed for use incombination with proprietary low-binding substrate surfaces and DNAamplification processes that reduce fluorescence background arising froma variety of confounding signals including, but are not limited to,nonspecific adsorption of fluorescent dyes to substrate surfaces,nonspecific nucleic acid amplification products (e.g., nucleic acidamplification products that arise the substrate surface in areas betweenthe spots or features corresponding to clonally-amplified clusters ofnucleic acid molecules (i.e., specifically amplified colonies),nonspecific nucleic acid amplification products that may arise withinthe amplified colonies, phased and pre-phased nucleic acid strands, etc.The use of low-binding substrate surfaces and DNA amplificationprocesses that reduce fluorescence background in combination with thedisclosed optical imaging systems may significantly cut down on the timerequired to image each FOV.

The presently disclosed system designs may further reduce the requiredimaging time through imaging sequence improvement or optimization wheremultiple channels of fluorescence images are acquired simultaneously orwith overlapping timing, and where spectral separation of thefluorescence signals is designed to reduce cross-talks betweenfluorescence detection channels and between the excitation light and thefluorescence signal(s).

The presently disclosed system designs may further reduce the requiredimaging time through improvement or optimization of scanning motionsequence. In the typical approach, an X-Y translation stage is used tomove the target FOV into position underneath the objective, an autofocusstep is performed where optimal focal position is determined and theobjective is moved in the Z direction to the determined focal position,and an image is acquired. A sequence of fluorescence images is acquiredby cycling through a series of target FOV positions. From an informationtransfer duty cycle perspective, information is only transferred duringthe fluorescence image acquisition portion of the cycle. In thepresently disclosed imaging system designs, a single-step motion inwhich all axes (X-Y-Z) are repositioned simultaneously is performed, andthe autofocus step is used to check focal position error. The additionalZ motion is only commanded if the focal position error (i.e., thedifference between the focal plane position and the sample planeposition) exceeds a certain limit (e.g., a specified error threshold).Coupled with high speed X-Y motion, this approach increases the dutycycle of the system, and thus increases the imaging throughput per unittime.

Furthermore, by matching the optical collection efficiency, modulationtransfer function, and image sensor performance characteristics of thedesign with the fluorescence photon flux expected for the inputexcitation photon flux, dye efficiency (related to dye extinctioncoefficient and fluorescence quantum yield), while accounting forbackground signal and system noise characteristics, the time required toacquire high quality (high contrast-to-noise ratio (CNR) images) may bereduced or minimized.

The combination of efficient image acquisition and improved or optimizedtranslation stage step and settle times leads to fast imaging times(i.e., the overall time required per field-of-view) and higherthroughput imaging system performance.

Along with the large FOV and fast image acquisition duty cycle, thedisclosed designs may comprise also specifying image plane flatness,chromatic focus performance between fluorescence detection channels,sensor flatness, image distortion, and focus quality specifications.

Chromatic focus performance is further improved by individually aligningthe image sensors for different fluorescence detection channels suchthat the best focal plane for each detection channel overlaps. Thedesign goal is to ensure that images across more than 90 percent of thefield-of-view are acquired within ±100 nm (or less) relative to the bestfocal plane for each channel, thus increasing or maximizing the transferof individual spot intensity signals. In some instances, the discloseddesigns further ensure that images across 99 percent of thefield-of-view are acquired within ±150 nm (or less) relative to the bestfocal plane for each channel, and that images across more the entirefield-of-view are acquired within ±200 nm (or less) relative to the bestfocal plane for each imaging channel.

Illumination optical path design: Another factor for improvingsignal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and/orincreasing throughput is to increase illumination power density to thesample. In some instances, the disclosed imaging systems may comprise anillumination path design that utilizes a high-power laser or laser diodecoupled with a liquid light guide. The liquid light guide removesoptical speckle that is intrinsic to coherent light sources such aslasers and laser diodes. Furthermore, the coupling optics are designedin such a way as to underfill the entrance aperture of the liquid lightguide. The underfilling of the liquid light guide entrance aperturereduces the effective numerical aperture of the illumination beamentering the objective lens, and thus improves light delivery efficiencythrough the objective onto the sample plane. With this designinnovation, one can achieve illumination power densities up to 3× thatfor conventional designs over a large field-of-view (FOV).

By utilizing the angle-dependent discrimination of s- andp-polarization, in some instances, the illumination beam polarizationmay be orientated to reduce the amount of back-scattered andback-reflected illumination light that reaches the imaging sensors.

Assessing image quality: For any of the embodiments of the opticalimaging designs disclosed herein, imaging performance or imaging qualitymay be assessed using any of a variety of performance metrics known tothose of skill in the art. Examples include, but are not limited to,measurements of modulation transfer function (MTF) at one or morespecified spatial frequencies, defocus, spherical aberration, chromaticaberration, coma, astigmatism, field curvature, image distortion,contrast-to-noise ratio (CNR), or any combination thereof.

In some instances, the disclosed optical designs for dual-side imaging(e.g., the disclosed objective lens designs, tube lens designs, the useof an electro-optical phase plate in combination with an objective,etc., alone or in combination) may yield significant improvements forimage quality for both the upper (near) and lower (far) interiorsurfaces of a flow cell, such that the difference in an imagingperformance metric for imaging the upper interior surface and the lowerinterior surface of the flow cell is less than 20%, less than 15%, lessthan 10%, less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% for any of the imaging performance metrics listed above,either individually or in combination.

In some instances, the disclosed optical designs for dual-side imaging(e.g., comprising the disclosed tube lens designs, the use of anelectro-optical phase plate in combination with an objective, etc.) mayyield significant improvements for image quality such that an imagequality performance metric for dual-side imaging provides for an atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, or at least 30%improvement in the imaging performance metric for dual-side imagingcompared to that for a conventional system comprising, e.g., anobjective lens, a motion-actuated compensator (that is moved out of orinto the optical path when imaging the near or far interior surfaces ofa flow cell), and an image sensor for any of the imaging performancemetrics listed above, either individually or in combination. In someinstances, fluorescence imaging systems comprising one or more of thedisclosed tube lens designs provides for an at least equivalent orbetter improvement in an imaging performance metric for dual-sideimaging compared to that for a conventional system comprising anobjective lens, a motion-actuated compensator, and an image sensor. Insome instances, fluorescence imaging systems comprising one or more ofthe disclosed tube lens designs provides for an at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% improvement in an imagingperformance metric for dual-side imaging compared to that for aconventional system comprising an objective lens, a motion-actuatedcompensator, and an image sensor.

Imaging Module Specifications:

Excitation light wavelength(s): In any of the disclosed optical imagingmodule designs, the light source(s) of the disclosed imaging modules mayproduce visible light, such as green light and/or red light. In someinstances, the light source(s), alone or in combination with one or moreoptical components, e.g., excitation optical filters and/or dichroicbeam splitters, may produce excitation light at about 350 nm, 375 nm,400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm,625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm,850 nm, 875 nm, or 900 nm. Those of skill in the art will recognize thatthe excitation wavelength may have any value within this range, e.g.,about 620 nm.

Excitation light bandwidths: In any of the disclosed optical imagingmodule designs, the light source(s), alone or in combination with one ormore optical components, e.g., excitation optical filters and/ordichroic beam splitters, may produce light at the specified excitationwavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm,±80 nm, or greater. Those of skill in the art will recognize that theexcitation bandwidths may have any value within this range, e.g., about±18 nm.

Light source power output: In any of the disclosed optical imagingmodule designs, the output of the light source(s) and/or an excitationlight beam derived therefrom (including a composite excitation lightbeam) may range in power from about 0.5 W to about 5.0 W, or more (aswill be discussed in more detail below). In some instances, the outputof the light source and/or the power of an excitation light beam derivedtherefrom may be at least 0.5 W, at least 0.6 W, at least 0.7 W, atleast 0.8 W, at least 1 W, at least 1.1 W, at least 1.2 W, at least 1.3W, at least 1.4 W, at least 1.5 W, at least 1.6 W, at least 1.8 W, atleast 2.0 W, at least 2.2 W, at least 2.4 W, at least 2.6 W, at least2.8 W, at least 3.0 W, at least 3.5 W, at least 4.0 W, at least 4.5 W,or at least 5.0 W. In some implementations, the output of the lightsource and/or the power of an excitation light beam derived therefrom(including a composite excitation light beam) may be at most 5.0 W, atmost 4.5 W, at most 4.0 W, at most 3.5 W, at most 3.0 W, at most 2.8 W,at most 2.6 W, at most 2.4 W, at most 2.2 W, at most 2.0 W, at most 1.8W, at most 1.6 W, at most 1.5 W, at most 1.4 W, at most 1.3 W, at most1.2 W, at most 1.1 W, at most 1 W, at most 0.8 W, at most 0.7 W, at most0.6 W, or at most 0.5 W. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the output of thelight source and/or the power of an excitation light beam derivedtherefrom (including a composite excitation light beam) may range fromabout 0.8 W to about 2.4 W. Those of skill in the art will recognizethat the output of the light source and/or the power of an excitationlight beam derived therefrom (including a composite excitation lightbeam) may have any value within this range, e.g., about 1.28 W.

Light source output power and CNR: In some implementations of thedisclosed optical imaging module designs, the output power of the lightsource(s) and/or the power of excitation light beam(s) derived therefrom(including a composite excitation light beam) is sufficient, incombination with an appropriate sample, to provide for acontrast-to-noise ratio (CNR) in images acquired by the illumination andimaging module of at least 5, at least 10, at least 15, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least30, at least 35, at least 40, or at least 50 or more, or any CNR withinany range formed by any of these values.

Fluorescence emission bands: In some instances, the disclosedfluorescence optical imaging modules may be configured to detectfluorescence emission produced by any of a variety of fluorophores knownto those of skill in the art. Examples of suitable fluorescence dyes foruse in, e.g., genotyping and nucleic acid sequencing applications (e.g.,by conjugation to nucleotides, oligonucleotides, or proteins) include,but are not limited to, fluorescein, rhodamine, coumarin, cyanine, andderivatives thereof, including the cyanine derivatives cyanine dye-3(Cy3), cyanine dye-5 (Cy5), cyanine dye-7 (Cy7), etc.

Fluorescence emission wavelengths: In any of the disclosed opticalimaging module designs, the detection channel or imaging channel of thedisclosed optical systems may include one or more optical components,e.g., emission optical filters and/or dichroic beam splitters,configured to collect emission light at about 350 nm, 375 nm, 400 nm,425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm,650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm,875 nm, or 900 nm. Those of skill in the art will recognize that theemission wavelength may have any value within this range, e.g., about825 nm.

Fluorescence emission light bandwidths: In any of the disclosed opticalimaging module designs, the detection channel or imaging channel maycomprise one or more optical components, e.g., emission optical filtersand/or dichroic beam splitters, configured to collect light at thespecified emission wavelength within a bandwidth of ±2 nm, ±5 nm, ±10nm, ±20 nm, ±40 nm, ±80 nm, or greater. Those of skill in the art willrecognize that the excitation bandwidths may have any value within thisrange, e.g., about ±18 nm.

Numerical aperture: In some instances, the numerical aperture of theobjective lens and/or optical imaging module (e.g., comprising anobjective lens and/or tube lens) in any of the disclosed optical systemdesigns may range from about 0.1 to about 1.4. In some instances, thenumerical aperture may be at least 0.1, at least 0.2, at least 0.3, atleast 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, atleast 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or atleast 1.4. In some instances, the numerical aperture may be at most 1.4,at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, atmost 0.2, or at most 0.1. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the numericalaperture may range from about 0.1 to about 0.6. Those of skill in theart will recognize that the numerical aperture may have any value withinthis range, e.g., about 0.55.

Optical resolution: In some instances, depending on the numericalaperture of the objective lens and/or optical system (e.g., comprisingan objective lens and/or tube lens), the minimum resolvable spot (orfeature) separation distance at the sample plane achieved by any of thedisclosed optical system designs may range from about 0.5 μm to about 2μm. In some instances, the minimum resolvable spot separation distanceat the sample plane may be at least 0.5 μm, at least 0.6 μm, at least0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 1.0μm. In some instances, the minimum resolvable spot separation distancemay be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm,at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe minimum resolvable spot separation distance may range from about 0.8μm to about 1.6 μm. Those of skill in the art will recognize that theminimum resolvable spot separation distance may have any value withinthis range, e.g., about 0.95 μm.

Optical resolution of first and second surfaces at different depths: Insome instances, the use of the novel objective lens and/or tube lensdesigns disclosed herein, in any of the optical modules or systemsdisclosed herein, may confer comparable optical resolution for first andsecond surfaces (e.g. the upper and lower interior surfaces of a flowcell) with or without the need to refocus between acquiring the imagesof the first and second surfaces. In some instances, the opticalresolution of the images thus obtained of the first and second surfacesmay be with 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of eachother, or within any value within this range.

Magnification: In some instances, the magnification of the objectivelens and/or tube lens, and/or optical system (e.g., comprising anobjective lens and/or tube lens) in any of the disclosed opticalconfigurations may range from about 2× to about 20×. In some instances,the optical system magnification may be at least 2×, at least 3×, atleast 4×, at least 5×, at least 6×, at least 7×, at least 8×, at least9×, at least 10×, at least 15×, or at least 20×. In some instances, theoptical system magnification may be at most 20×, at most 15×, at most10×, at most 9×, at most 8×, at most 7×, at most 6×, at most 5×, at most4×, at most 3×, or at most 2×. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances theoptical system magnification may range from about 3× to about 10×. Thoseof skill in the art will recognize that the optical system magnificationmay have any value within this range, e.g., about 7.5×.

Objective lens focal length: In some implementations of the disclosedoptical designs, the focal length of the objective lens may rangebetween 20 mm and 40 mm. In some instances, the focal length of theobjective lens may be at least 20 mm, at least 25 mm, at least 30 mm, atleast 35 mm, or at least 40 mm. In some instances, the focal length ofthe objective lens may be at most 40 mm, at most 35 mm, at most 30 mm,at most 25 mm, or at most 20 mm. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the focallength of the objective lens may range from 25 mm to 35 mm. Those ofskill in the art will recognize that the focal length of the objectivelens may have any value within the range of values specified above,e.g., about 37 mm.

Objective lens working distance: In some implementations of thedisclosed optical designs, the working distance of the objective lensmay range between about 100 μm and 30 mm. In some instances, the workingdistance may be at least 100 μm, at least 200 μm, at least 300 μm, atleast 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, atleast 800 μm, at least 900 μm, at least 1 mm, at least 2 mm, at least 4mm, at least 6 mm, at least 8 mm, at least 10 mm, at least 15 mm, atleast 20 mm, at least 25 mm, or at least 30 mm. In some instances, theworking distance may be at most 30 mm, at most 25 mm, at most 20 mm, atmost 15 mm, at most 10 mm, at most 8 mm, at most 6 mm, at most 4 mm, atmost 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm,at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most200 μm, at most 100 μm. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the working distanceof the objective lens may range from 500 μm to 2 mm. Those of skill inthe art will recognize that the working distance of the objective lensmay have any value within the range of values specified above, e.g.,about 1.25 mm.

Objectives optimized for imaging through thick coverslips: In someinstances of the disclosed optical designs, the design of the objectivelens may be improved or optimized for a different coverslip of flow cellthickness. For example, in some instances the objective lens may bedesigned for optimal optical performance for a coverslip that is fromabout 200 μm to about 1,000 μm thick. In some instances, the objectivelens may be designed for optimal performance with a coverslip that is atleast 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, atleast 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or atleast 1,000 μm thick. In some instances, the objective lens may bedesigned for optimal performance with a coverslip that is at most 1,000μm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, atmost 500 μm, at most 400 μm, at most 300 μm, or at most 200 μm thick.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the objective lens may be designed foroptimal optical performance for a coverslip that may range from about300 μm to about 900 μm. Those of skill in the art will recognize thatthe objective lens may be designed for optimal optical performance for acoverslip that may have any value within this range, e.g., about 725 μm.

Depth of field and depth of focus: In some instances, the depth of fieldand/or depth of focus for any of the disclosed imaging module (e.g.,comprising an objective lens and/or tube lens) designs may range fromabout 10 μm to about 800 μm, or more. In some instances, the depth offield and/or depth of focus may be at least 10 μm, at least 20 μm, atleast 30 μm, at least 40 μm, at least 50 μm, at least 75 μm, at least100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200μm, at least 250 μm, at least 300 μm, at least 300 μm, at least 400 μm,at least 500 μm, at least 600 μm, at least 700 μm, or at least 800 μm,or more. In some instances, the depth of field and/or depth of focus beat most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most400 μm, at most 300 μm, at most 250 μm, at most 200 μm, at most 175 μm,at most 150 μm, at most 125 μm, at most 100 μm, at most 75 μm, at most50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, orless. Any of the lower and upper values described in this paragraph maybe combined to form a range included within the present disclosure, forexample, in some instances the depth of field and/or depth of focus mayrange from about 100 μm to about 175 μm. Those of skill in the art willrecognize that the depth of field and/or depth of focus may have anyvalue within the range of values specified above, e.g., about 132 μm.

Field of view (FOV): In some implementations, the FOV of any of thedisclosed imaging module designs (e.g., that provided by a combinationof objective lens and detection channel optics (such as a tube lens))may range, for example, between about 1 mm and 5 mm (e.g., in diameter,width, length, or longest dimension). In some instances, the FOV may beat least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, atleast 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, or atleast 5.0 mm (e.g., in diameter, width, length, or longest dimension).In some instances, the FOV may be at most 5.0 mm, at most 4.5 mm, atmost 4.0 mm, at most 3.5 mm, at most 3.0 mm, at most 2.5 mm, at most 2.0mm, at most 1.5 mm, or at most 1.0 mm (e.g., in diameter, width, length,or longest dimension). Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the FOV may rangefrom about 1.5 mm to about 3.5 mm (e.g., in diameter, width, length, orlongest dimension). Those of skill in the art will recognize that theFOV may have any value within the range of values specified above, e.g.,about 3.2 mm (e.g., in diameter, width, length, or longest dimension).

Field-of-view (FOV) area: In some instances of the disclosed opticalsystem designs, the area of the field-of-view may range from about 2 mm²to about 5 mm². In some instances, the field-of-view may be at least 2mm², at least 3 mm², at least 4 mm², or at least 5 mm² in area. In someinstances, the field-of-view may be at most 5 mm², at most 4 mm², atmost 3 mm², or at most 2 mm² in area. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thefield-of-view may range from about 3 mm² to about 4 mm² in area. Thoseof skill in the art will recognize that the area of the field-of-viewmay have any value within this range, e.g., 2.75 mm².

Optimization of objective lens and/or tube lens MTF: In some instances,the design of the objective lens and/or at least one tube lens in thedisclosed imaging modules and systems is configured to optimize themodulation transfer function in the mid to high spatial frequency range.For example, in some instances, the design of the objective lens and/orat least one tube lens in the disclosed imaging modules and systems isconfigured to optimize the modulation transfer function in the spatialfrequency range from 500 cycles per mm to 900 cycles per mm, from 700cycles per mm to 1100 cycles per mm, from 800 cycles per mm to 1200cycles per mm, or from 600 cycles per mm to 1000 cycles per mm in thesample plane.

Optical aberration and diffraction-limited imaging performance: In someimplementations of any of the optical imaging module designs disclosedherein, the objective lens and/or tube lens may be configured to providethe imaging module with a field-of-view as indicated above such that theFOV has less than 0.15 waves of aberration over at least 60%, 70%, 80%,90%, or 95% of the field. In some implementations, the objective lensand/or tube lens may be configured to provide the imaging module with afield-of-view as indicated above such that the FOV has less than 0.1waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of thefield. In some implementations, the objective lens and/or tube lens maybe configured to provide the imaging module with a field-of-view asindicated above such that the FOV has less than 0.075 waves ofaberration over at least 60%, 70%, 80%, 90%, or 95% of the field. Insome implementations, the objective lens and/or tube lens may beconfigured to provide the imaging module with a field-of-view asindicated above such that the FOV is diffraction-limited over at least60%, 70%, 80%, 90%, or 95% of the field.

Angle of incidence of light beams on dichroic reflectors, beam splitter,and beam combiners: In some instances of the disclosed optical designs,the angles of incidence for a light beam incident on a dichroicreflector, beam splitter, or beam combiner may range between about 20degrees and about 45 degrees. In some instances, the angles of incidencemay be at least 20 degrees, at least 25 degrees, at least 30 degrees, atleast 35 degrees, at least 40 degrees, or at least 45 degrees. In someinstances, the angles of incidence may be at most 45 degrees, at most 40degrees, at most 35 degrees, at most 30 degrees, at most 25 degrees, orat most 20 degrees. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the angles of incidence mayrange from about 25 degrees to about 40 degrees. Those of skill in theart will recognize that the angles of incidence may have any valuewithin the range of values specified above, e.g., about 43 degrees.

Image sensor (photodetector array) size: In some instances, thedisclosed optical systems may comprise image sensor(s) having an activearea with a diagonal ranging from about 10 mm to about 30 mm, or larger.In some instances, the image sensors may have an active area with adiagonal of at least 10 mm, at least 12 mm, at least 14 mm, at least 16mm, at least 18 mm, at least 20 mm, at least 22 mm, at least 24 mm, atleast 26 mm, at least 28 mm, or at least 30 mm. In some instances, theimage sensors may have an active area with a diagonal of at most 30 mm,at most 28 mm, at most 26 mm, at most 24 mm, at most 22 mm, at most 20mm, at most 18 mm, at most 16 mm, at most 14 mm, at most 12 mm, or atmost 10 mm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the image sensor(s) may havean active area with a diagonal ranging from about 12 mm to about 24 mm.Those of skill in the art will recognize that the image sensor(s) mayhave an active area with a diagonal having any value within the range ofvalues specified above, e.g., about 28.5 mm.

Image sensor pixel size and pitch: In some instances, the pixel sizeand/or pitch selected for the image sensor(s) used in the disclosedoptical system designs may range in at least one dimension from about 1μm to about 10 μm. In some instances, the pixel size and/or pitch may beat least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or atleast 10 μm. In some instances, the pixel size and/or pitch may be atmost 10 μm, at most 9 μm, at most 8 μm, at most 7 μm, at most 6 μm, atmost 5 μm, at most 4 μm, at most 3 μm, at most 2 μm, or at most 1 μm.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the pixel size and/or pitch may range fromabout 3 μm to about 9 μm. Those of skill in the art will recognize thatthe pixel size and/or pitch may have any value within this range, e.g.,about 1.4 μm.

Oversampling: In some instances of the disclosed optical designs, aspatial oversampling scheme is utilized wherein the spatial samplingfrequency is at least 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×,or 10× the optical resolution X (lp/mm).

Maximum translation stage velocity: In some instances of the disclosedoptical imaging modules, the maximum translation stage velocity on anyone axis may range from about 1 mm/sec to about 5 mm/sec. In someinstances, the maximum translation stage velocity may be at least 1mm/sec, at least 2 mm/sec, at least 3 mm/sec, at least 4 mm/sec, or atleast 5 mm/sec. In some instances, the maximum translation stagevelocity may be at most 5 mm/sec, at most 4 mm/sec, at most 3 mm/sec, atmost 2 mm/sec, or at most 1 mm/sec. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances themaximum translation stage velocity may range from about 2 mm/sec toabout 4 mm/sec. Those of skill in the art will recognize that themaximum translation stage velocity may have any value within this range,e.g., about 2.6 mm/sec.

Maximum translation stage acceleration: In some instances of thedisclosed optical imaging modules, the maximum acceleration on any oneaxis of motion may range from about 2 mm/sec² to about 10 mm/sec². Insome instances, the maximum acceleration may be at least 2 mm/sec², atleast 3 mm/sec², at least 4 mm/sec², at least 5 mm/sec², at least 6mm/sec², at least 7 mm/sec², at least 8 mm/sec², at least 9 mm/sec², orat least 10 mm/sec². In some instances, the maximum acceleration may beat most 10 mm/sec², at most 9 mm/sec², at most 8 mm/sec², at most 7mm/sec², at most 6 mm/sec², at most 5 mm/sec², at most 4 mm/sec², atmost 3 mm/sec², or at most 2 mm/sec². Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances themaximum acceleration may range from about 2 mm/sec² to about 8 mm/sec².Those of skill in the art will recognize that the maximum accelerationmay have any value within this range, e.g., about 3.7 mm/sec².

Translation stage positioning repeatability: In some instances of thedisclosed optical imaging modules, the repeatability of positioning forany one axis may range from about 0.1 μm to about 2 In some instances,the repeatability of positioning may be at least 0.1 μm, at least 0.2μm, at least 0.3 μm, at least 0.4 μm, at least 0.5 μm, at least 0.6 μm,at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, atleast 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or atleast 2.0 μm. In some instances, the repeatability of positioning may beat most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm,at most 0.6 μm, at most 0.5 μm, at most 0.4 μm, at most 0.3 μm, at most0.2 μm, or at most 0.1 μm. Any of the lower and upper values describedin this paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the repeatability ofpositioning may range from about 0.3 μm to about 1.2 μm. Those of skillin the art will recognize that the repeatability of positioning may haveany value within this range, e.g., about 0.47 μm.

FOV repositioning time: In some instances of the disclosed opticalimaging modules, the maximum time required to reposition the sampleplane (field-of-view) relative to the optics, or vice versa, may rangefrom about 0.1 sec to about 0.5 sec. In some instances, the maximumrepositioning time (i.e., the scan stage step and settle time) may be atleast 0.1 sec, at least 0.2 sec, at least 0.3 sec, at least 0.4 sec, orat least 0.5 sec. In some instances, the maximum repositioning time maybe at most 0.5 sec, at most 0.4 sec, at most 0.3 sec, at most 0.2 sec,or at most 0.1 sec. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the maximum repositioningtime may range from about 0.2 sec to about 0.4 sec. Those of skill inthe art will recognize that the maximum repositioning time may have anyvalue within this range, e.g., about 0.45 sec.

Error threshold for autofocus correction: In some instances of thedisclosed optical imaging modules, the specified error threshold fortriggering an autofocus correction may range from about 50 nm to about200 nm. In some instances, the error threshold may be at least 50 nm, atleast 75 nm, at least 100 nm, at least 125 nm, at least 150 nm, at least175 nm, or at least 200 nm. In some instances, the error threshold maybe at most 200 nm, at most 175 nm, at most 150 nm, at most 125 nm, atmost 100 nm, at most 75 nm, or at most 50 nm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe error threshold may range from about 75 nm to about 150 nm. Those ofskill in the art will recognize that the error threshold may have anyvalue within this range, e.g., about 105 nm.

Image acquisition time: In some instances of the disclosed opticalimaging modules, the image acquisition time may range from about 0.001sec to about 1 sec. In some instances, the image acquisition time may beat least 0.001 sec, at least 0.01 sec, at least 0.1 sec, or at least 1sec. in some instances, the image acquisition time may be at most 1 sec,at most 0.1 sec, at most 0.01 sec, or at most 0.001 sec. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the image acquisition time may range from about 0.01 secto about 0.1 sec. Those of skill in the art will recognize that theimage acquisition time may have any value within this range, e.g., about0.250 seconds.

Imaging time per FOV: In some instances, the imaging times may rangefrom about 0.5 seconds to about 3 seconds per field-of-view. In someinstances, the imaging time may be at least 0.5 seconds, at least 1second, at least 1.5 seconds, at least 2 seconds, at least 2.5 seconds,or at least 3 seconds per FOV. In some instances, the imaging time maybe at most 3 seconds, at most 2.5 seconds, at most 2 seconds, at most1.5 seconds, at most 1 second, or at most 0.5 seconds per FOV. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the imaging time may range from about 1 second to about2.5 seconds. Those of skill in the art will recognize that the imagingtime may have any value within this range, e.g., about 1.85 seconds.

Flatness of field: In some instances, images across 80%, 90%, 95%, 98%,99%, or 100% percent of the field-of-view are acquired within ±200 nm,±175 nm, ±150 nm, ±125 nm, ±100 nm, ±75 nm, or ±50 nm relative to thebest focal plane for each fluorescence (or other imaging mode) detectionchannel.

Analysis systems and system components for genomics and otherapplications: As noted above, in some implementations, the disclosedoptical imaging modules may function as modules, components,sub-assemblies, or sub-systems of larger systems (e.g., analysissystems) configured for performing, e.g., genomics applications (e.g.,genetic testing and/or nucleic acid sequencing applications) or otherchemical analysis, biochemical analysis, nucleic acid analysis, cellanalysis or tissue analysis applications. In addition to one, two,three, four, or more than four imaging modules as disclosed herein (eachof which may comprise one or more illumination optical paths and/or oneor more detection optical paths (e.g., one or more detection channelsconfigured for imaging fluorescence emission within a specifiedwavelength range onto an image sensor)), such systems may comprise oneor more X-Y translation stages, one or more X-Y-Z translation stages,flow cells or cartridges, fluidics systems and fluid flow controlmodules, temperature control modules, fluid dispensing robotics,cartridge- and/or microplate-handling (pick-and-place) robotics,light-tight housings and/or environmental control chambers, one or moreprocessors or computers, data storage modules, data communicationmodules (e.g., Bluetooth, WiFi, intranet, or internet communicationhardware and associated software), display modules, one or more localand/or cloud-based software packages (e.g., instrument/system controlsoftware packages, image processing software packages, data analysissoftware packages), etc., or any combination thereof.

Translation stages: In some implementations of the imaging and analysissystems (e.g., nucleic acid sequencing systems) disclosed herein, thesystem may comprise one or more (e.g., one, two, three, four, or morethan four) high precision X-Y (or in some cases, X-Y-Z) translationstage(s) for re-positioning one or more sample support structure(s)(e.g., flow cell(s)) in relation to the one or more imaging modules, forexample, in order to tile one or more images, each corresponding to afield-of-view of the imaging module, to reconstruct composite image(s)of an entire flow cell surface. In some implementations of the imagingsystems and genomics analysis systems (e.g., nucleic acid sequencingsystems) disclosed herein, the system may comprise one or more (e.g.,one, two, three, four, or more than four) high precision X-Y (or in somecases, X-Y-Z) translation stage(s) for re-positioning the one or moreimaging modules in relation to one or more sample support structure(s)(e.g., flow cell(s)), for example, in order to tile one or more images,each corresponding to a field-of-view of the imaging module, toreconstruct composite image(s) of an entire flow cell surface.

Suitable translation stages are commercially available from a variety ofvendors, for example, Parker Hannifin. Precision translation stagesystems typically comprise a combination of several componentsincluding, but not limited to, linear actuators, optical encoders, servoand/or stepper motors, and motor controllers or drive units. Highprecision and repeatability of stage movement is required for thesystems and methods disclosed herein in order to ensure accurate andreproducible positioning and imaging of, e.g., fluorescence signals wheninterspersing repeated steps of reagent delivery and optical detection.

Consequently, the systems disclosed herein may comprise specifying theprecision with which the translation stage is configured to position asample support structure in relation to the illumination and/or imagingoptics (or vice versa). In one aspect of the present disclosure, theprecision of the one or more translation stages is between about 0.1 μmand about 10 μm. In other aspects, the precision of the translationstage is about 10 μm or less, about 9 μm or less, about 8 μm or less,about 7 μm or less, about 6 μm or less, about 5 μm or less, about 4 μmor less, about 3 μm or less, about 2 μm or less, about 1 μm or less,about 0.9 μm or less, about 0.8 μm or less, about 0.7 μm or less, about0.6 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μmor less, about 0.2 μm or less, or about 0.1 μm or less. Those of skillin the art will appreciate that, in some instances, the positioningprecision of the translation stage may fall within any range bounded byany of two of these values (e.g. from about 0.5 μm to about 1.5 μm). Insome instances, the positioning precision of the translation stage mayhave any value within the range of values included in this paragraph,e.g., about 0.12 μm.

Flow cells, microfluidic devices, and cartridges: As noted above, insome instances, a sample support structure for the disclosed imagingmodules may be configured as a flow cell device comprising, e.g., one,two, three, four, or more than four sample support surfaces (or simplysurfaces) upon which cells, tissue slices, or nucleic acid moleculesderived therefrom may be tethered or immobilized. The flow cell devicesand flow cell cartridges disclosed herein may be used as components ofanalysis systems designed for a variety of chemical analysis,biochemical analysis, nucleic acid analysis, cell analysis, or tissueanalysis application. In general, such analysis systems may comprise oneor more one or more of the disclosed single capillary flow cell devices,multiple capillary flow cell devices, capillary flow cell cartridges,and/or microfluidic devices and cartridges described herein. Additionaldescription of the disclosed flow cell devices and cartridges may befound in PCT Patent Application Publication WO 2020/118255, which isincorporated herein by reference in its entirety.

In some instances, the systems disclosed herein may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 single capillary flow cell devices,multiple capillary flow cell devices, capillary flow cell cartridges,and/or microfluidic devices and cartridges. In some instances, thesingle capillary flow cell devices, multiple capillary flow celldevices, and/or microfluidic devices and cartridges may be fixedcomponents of the disclosed systems. In some instances, the singlecapillary flow cell devices, multiple capillary flow cell devices,and/or microfluidic devices and cartridges may be removable,exchangeable components of the disclosed systems. In some instances, thesingle capillary flow cell devices, multiple capillary flow celldevices, and/or microfluidic devices and cartridges may be disposable orconsumable components of the disclosed systems.

In some implementations, the disclosed single capillary flow celldevices (or single capillary flow cell cartridges) comprise a singlecapillary, e.g., a glass or fused-silica capillary, the lumen of whichforms a fluid flow path through which reagents or solutions may flow,and the interior surface of which may form a sample support structure towhich samples of interest are bound or tethered. In someimplementations, the multi-capillary capillary flow cell devices (ormulti-capillary flow cell cartridges) disclosed herein may comprise 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morethan 20 capillaries configured for performing an analysis technique thatfurther comprises imaging as a detection method.

In some instances, one or more capillaries may be packaged within achassis to form a cartridge that facilitates ease-of-handling,incorporates adapters or connectors for making external fluidconnections, and may optionally include additional integratedfunctionality such as reagent reservoirs, waste reservoirs, valves(e.g., microvalves), pumps (e.g., micropumps), etc., or any combinationthereof.

FIG. 24 illustrates one non-limiting example of a single glass capillaryflow cell device that comprises two fluidic adaptors—one affixed to eachend of the piece of glass capillary—that are designed to mate withstandard OD fluidic tubing to provide for convenient, interchangeablefluid connections with an external fluid flow control system. Thefluidic adaptors can be attached to the capillary using any of a varietyof techniques known to those of skill in the art including, but notlimited to, press fit, adhesive bonding, solvent bonding, laser welding,etc., or any combination thereof.

In general, the capillaries used in the disclosed capillary flow celldevices and capillary flow cell cartridges will have at least oneinternal, axially-aligned fluid flow channel (or “lumen”) that runs thefull length of the capillary. In some instances, the capillary may havetwo, three, four, five, or more than five internal, axially-alignedfluid flow channels (or “lumen”).

A number specified cross-sectional geometries for suitable capillaries(or the lumen thereof) are consistent with the disclosure hereinincluding, but not limited to, circular, elliptical, square,rectangular, triangular, rounded square, rounded rectangular, or roundedtriangular cross-sectional geometries. In some instances, the capillary(or lumen thereof) may have any specified cross-sectional dimension orset of dimensions. For example, in some instances the largestcross-sectional dimension of the capillary lumen (e.g. the diameter ifthe lumen is circular in shape, or the diagonal if the lumen is squareor rectangular in shape) may range from about 10 μm to about 10 mm. Insome aspects, the largest cross-sectional dimension of the capillarylumen may be at least 10 μm, at least 25 μm, at least 50 μm, at least 75μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm,at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, atleast 900 μm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least9 mm, or at least 10 mm. In some aspects, the largest cross-sectionaldimension of the capillary lumen may be at most 10 mm, at most 9 mm, atmost 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, atmost 3 mm, at most 2 mm, at most 1 mm, at most 900 μm, at most 800 μm,at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, atmost 25 μm, or at most 10 μm. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thelargest cross-sectional dimension of the capillary lumen may range fromabout 100 μm to about 500 μm. Those of skill in the art will recognizethat the largest cross-sectional dimension of the capillary lumen mayhave any value within this range, e.g., about 124 μm.

In some instances, e.g., wherein the lumen of the one or morecapillaries in a flow cell device or cartridge has a square orrectangular cross-section, the distance between a first interior surface(e.g., a top or upper surface) and a second interior surface (e.g., abottom or lower surface) that defines the gap height or thickness of afluid flow channel may range from about 10 μm to about 500 μm. In someinstances, the gap height may be at least 10 μm, at least 20 μm, atleast 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, atleast 150 μm, at least 175 μm, at least 200 μm, at least 225 μm, atleast 250 μm, at least 275 μm, at least 300 μm, at least 325 μm, atleast 350 μm, at least 375 μm, at least 400 μm, at least 425 μm, atleast 450 μm, at least 475 μm, or at least 500 μm. In some instances,the gap height may be at most 500 μm, at most 475 μm, at most 450 μm, atmost 425 μm, at most 400 μm, at most 375 μm, at most 350 μm, at most 325μm, at most 300 μm, at most 275 μm, at most 250 μm, at most 225 μm, atmost 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most50 μm, at most 40 μm, at most 30 μm, at most 20 μm, or most 10 μm. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the gap height may range from about 40 μm toabout 125 μm. Those of skill in the art will recognize that the gapheight may have any value within the range of values in this paragraph,e.g., about 122 μm.

In some instances, the length of the one or more capillaries used tofabricate the disclosed capillary flow cell devices or flow cellcartridges may range from about 5 mm to about 5 cm or greater. In someinstances, the length of the one or more capillaries may be less than 5mm, at least 5 mm, at least 1 cm, at least 1.5 cm, at least 2 cm, atleast 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least4.5 cm, or at least 5 cm. In some instances, the length of the one ormore capillaries may be at most 5 cm, at most 4.5 cm, at most 4 cm, atmost 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, at most 1.5 cm,at most 1 cm, or at most 5 mm. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the lengthof the one or more capillaries may range from about 1.5 cm to about 2.5cm. Those of skill in the art will recognize that the length of the oneor more capillaries may have any value within this range, e.g., about1.85 cm. In some instances, devices or cartridges may comprise aplurality of two or more capillaries that are the same length. In someinstances, devices or cartridges may comprise a plurality of two or morecapillaries that are of different lengths.

The capillaries used for constructing the disclosed capillary flow celldevices or capillary flow cell cartridges may be fabricated from any ofa variety of materials known to those of skill in the art including, butnot limited to, glass (e.g., borosilicate glass, soda lime glass, etc.),fused silica (quartz), polymer (e.g., polystyrene (PS), macroporouspolystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC),polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),cyclic olefin polymers (COP), cyclic olefin copolymers (COC),polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.),polyetherimide (PEI) and perfluoroelastomer (FFKM) as more chemicallyinert alternatives, or any combination thereof. PEI is somewhere betweenpolycarbonate and PEEK in terms of both cost and chemical compatibility.FFKM is also known as Kalrez.

The one or more materials used to fabricate the capillaries are oftenoptically transparent to facilitate use with spectroscopic orimaging-based detection techniques. In some instances, the entirecapillary will be optically transparent. Alternately, in some instances,only a portion of the capillary (e.g., an optically transparent“window”) will be optically transparent.

The capillaries used for constructing the disclosed capillary flow celldevices and capillary flow cell cartridges may be fabricated using anyof a variety of techniques known to those of skill in the art, where thechoice of fabrication technique is often dependent on the choice ofmaterial used, and vice versa. Examples of suitable capillaryfabrication techniques include, but are not limited to, extrusion,drawing, precision computer numerical control (CNC) machining andboring, laser photoablation, and the like.

In some implementations, the capillaries used in the disclosed capillaryflow cell devices and cartridges may be off-the-shelf commercialproducts. Examples of commercial vendors that provide precisioncapillary tubing include Accu-Glass (St. Louis, Mo.; precision glasscapillary tubing), Polymicro Technologies (Phoenix, Ariz.; precisionglass and fused-silica capillary tubing), Friedrich & Dimmock, Inc.(Millville, N.J.; custom precision glass capillary tubing), and DrummondScientific (Broomall, Pa.; OEM glass and plastic capillary tubing).

The fluidic adapters that are attached to the capillaries of thecapillary flow cell devices and cartridges disclosed herein, and othercomponents of the capillary flow cell devices or cartridges, may befabricated using any of a variety of suitable techniques (e.g.,extrusion molding, injection molding, compression molding, precision CNCmachining, etc.) and materials (e.g., glass, fused-silica, ceramic,metal, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET), etc.), where again the choice of fabricationtechnique is often dependent on the choice of material used, and viceversa.

FIG. 25 provides a non-limiting example of capillary flow cell cartridgethat comprises two glass capillaries, fluidic adaptors (two percapillary in this example), and a cartridge chassis that mates with thecapillaries and/or fluidic adapters such that the capillaries are heldin a fixed orientation relative to the cartridge. In some instances, thefluidic adaptors may be integrated with the cartridge chassis. In someinstances, the cartridge may comprise additional adapters that mate withthe capillaries and/or capillary fluidic adapters. As noted elsewhereherein, in some instances, the cartridge may comprise additionalfunctional components. In some instances, the capillaries arepermanently mounted in the cartridge. In some instances, the cartridgechassis is designed to allow one or more capillaries of the flow cellcartridge to be interchangeable removed and replaced. For example, insome instances, the cartridge chassis may comprise a hinged “clamshell”configuration which allows it to be opened so that one or morecapillaries may be removed and replaces. In some instances, thecartridge chassis is configured to mount on, for example, the stage of afluorescence microscope or within a cartridge holder of a fluorescenceimaging module or instrument system of the present disclosure.

In some instances, the disclosed flow cell devices may comprisemicrofluidic devices (or “microfluidic chips”) and cartridges, where themicrofluidic devices are fabricated by forming fluid channels in one ormore layers of a suitable material and comprise one or more fluidchannels (e.g., “analysis” channels) configured for performing ananalysis technique that further comprises imaging as a detection method.In some implementations, the microfluidic devices or cartridgesdisclosed herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more than 20 fluid channels (e.g.,“analysis” fluid channels) configured for performing an analysistechnique that further comprises imaging as a detection method. In someinstances, the disclosed microfluidic devices may further compriseadditional fluid channels (e.g., for dilution or mixing of reagents),reagent reservoirs, waste reservoirs, adapters for making external fluidconnections, and the like, to provide integrated “lab-on-a-chip”functionality within the device.

A non-limiting example of microfluidic flow cell cartridge comprises achip having two or more parallel glass channels formed on the chip,fluidic adaptors coupled to the chip, and a cartridge chassis that mateswith the chip and/or fluidic adapters such that the chip is posited in afixed orientation relative to the cartridge. In some instances, thefluidic adaptors may be integrated with the cartridge chassis. In someinstances, the cartridge may comprise additional adapters that mate withthe chip and/or fluidic adapters. In some instances, the chip ispermanently mounted in the cartridge. In some instances, the cartridgechassis is designed to allow one or more chips of the flow cellcartridge to be interchangeably removed and replaced. For example, insome instances, the cartridge chassis may comprise a hinged “clamshell”configuration which allows it to be opened so that one or more chips maybe removed and replaces. In some instances, the cartridge chassis isconfigured to mount on, for example, the stage of a microscope system orwithin a cartridge holder of an imaging system. Even through only onechip is described in the non-limiting example, it is understood thatmore than one chip can be used in the microfluidic flow cell cartridge.The flow cell cartridges of the present disclosure may comprise a singlemicrofluidic chip or a plurality of microfluidic chips. In someinstances, the flow cell cartridges of the present disclosure maycomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more than 20 microfluidic chips. The packaging of one or moremicrofluidic devices within a cartridge may facilitate ease-of-handlingand correct positioning of the device within the optical imaging system.

The fluid channels within the disclosed microfluidic devices andcartridges may have an of a variety of cross-sectional geometriesincluding, but not limited to, circular, elliptical, square,rectangular, triangular, rounded square, rounded rectangular, or roundedtriangular cross-sectional geometries. In some instances, the fluidchannels may have any specified cross-sectional dimension or set ofdimensions. For example, in some instances, the height (e.g., gapheight), width, or largest cross-sectional dimension of the fluidchannels (e.g., the diagonal if the fluid channel has a square, roundedsquare, rectangular, or rounded rectangular cross-section) may rangefrom about 10 μm to about 10 mm. In some aspects, the height (e.g., gapheight), width, or largest cross-sectional dimension of the fluidchannels may be at least 10 μm, at least 25 μm, at least 50 μm, at least75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm,at least 900 μm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least9 mm, or at least 10 mm. In some aspects, the height (e.g., gap height),width, or largest cross-sectional dimension of the fluid channels may beat most 10 mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm,at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, atmost 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, atmost 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the height (e.g., gap height), width, or largestcross-sectional dimension of the fluid channels may range from about 20μm to about 200 μm. Those of skill in the art will recognize that theheight (e.g., gap height), width, or largest cross-sectional dimensionof the fluid channels may have any value within this range, e.g., about122 μm.

In some instances, the length of the fluid channels in the disclosedmicrofluidic devices and cartridges may range from about 5 mm to about10 cm or greater. In some instances, the length of the fluid channelsmay be less than 5 mm, at least 5 mm, at least 1 cm, at least 1.5 cm, atleast 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4cm, at least 4.5 cm, at least 5 cm, at least 6 cm, at least 7 cm, atleast 8 cm, at least 9 cm, or at least 10 cm. In some instances, thelength of the fluid channels may be at most 10 cm, at most 9 cm, at most8 cm, at most 7 cm, at most 6 cm, at most 5 cm, at most 4.5 cm, at most4 cm, at most 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, atmost 1.5 cm, at most 1 cm, or at most 5 mm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe length of the fluid channels may range from about 1.5 cm to about2.5 cm. Those of skill in the art will recognize that the length of thefluid channels may have any value within this range, e.g., about 1.35cm. In some instances, the microfluidic devices or cartridges maycomprise a plurality of fluid channels that are the same length. In someinstances, the microfluidic devices or cartridges may comprise aplurality of fluid channels that are of different lengths.

The disclosed microfluidic devices will comprise at least one layer ofmaterial having one or more fluid channels formed therein. In someinstances, the microfluidic chip may include two layers bonded togetherto form one or more fluid channels. In some instances, the microfluidicchip may include three or more layers bonded together to form one ormore fluid channels. In some instances, the microfluidic fluid channelsmay have an open top. In some instances, the microfluidic fluid channelsmay be fabricated within one layer, e.g., the top surface of a bottomlayer, and sealed by bonding the top surface of the bottom layer to thebottom surface of a top layer of material. In some instances, themicrofluidic channels may be fabricated within one layer, e.g., aspatterned channels the depth of which extends through the full thicknessof the layer, which is then sandwiched between and bonded to twonon-patterned layers to seal the fluid channels. In some instances, themicrofluidic channels are fabricated by the removal of a sacrificiallayer on the surface of a substrate. This method does not require thebulk substrate (e.g., a glass or silicon wafer) to be etched away.Instead, the fluid channels are located on the surface of the substrate.In some instances, the microfluidic channels may be fabricated in or onthe surface of a substrate and then sealed by deposition of a conformalfilm or layer on the surface of the substrate to create sub-surface orburied fluid channels in the chip.

The microfluidic chips can be manufactured using a combination ofmicrofabrication processes. Because the devices are microfabricated,substrate materials will typically be selected based upon theircompatibility with known microfabrication techniques, e.g.,photolithography, wet chemical etching, laser ablation, laserirradiation, air abrasion techniques, injection molding, embossing, andother techniques. The substrate materials are also generally selectedfor their compatibility with the full range of conditions to which themicrofluidic devices may be exposed, including extremes of pH,temperature, salt concentration, and application of electromagnetic(e.g. light) or electric fields.

The disclosed microfluidic chips may be fabricated from any of a varietyof materials known to those of skill in the art including, but notlimited to, glass (e.g., borosilicate glass, soda lime glass, etc.),fused-silica (quartz), silicon, a polymer (e.g., polystyrene (PS),macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),polycarbonate (PC), polypropylene (PP), polyethylene (PE), high densitypolyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefincopolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane(PDMS), etc.), polyetherimide (PEI) and perfluoroelastomer (FFKM) (asmore chemically inert alternatives), or any combination thereof. In somepreferred instances, the substrate material(s) may include silica-basedsubstrates, such as borosilicate glass, and quartz, as well as othersubstrate materials.

The disclosed microfluidic devices may be fabricated using any of avariety of techniques known to those of skill in the art, where thechoice of fabrication technique is often dependent on the choice ofmaterial used, and vice versa. The microfluidic channels on the chip canbe constructed using techniques suitable for forming micro-structures ormicro-patterns on the surface of a substrate. In some instances, thefluid channels are formed by laser irradiation. In some instances, themicrofluidic channels are formed by focused femtosecond laser radiation.In some instances, the microfluidic channels are formed byphotolithography and etching including, but not limited to, chemicaletching, plasma etching, or deep reactive ion etching. In someinstances, the microfluidic channels are formed using laser etching. Insome instances, the microfluidic channels are formed using adirect-write lithography technique. Examples of direct-write lithographyinclude electron beam direct-write and focused ion beam milling.

In additional preferred instances, the substrate material(s) maycomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, and the like. Such polymeric substrates may be readilypatterned or micromachined using available microfabrication techniques,such as those described above. In some instances, microfluidic chips maybe fabricated from polymeric materials, e.g., from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing, stamping, or by polymerizing the polymeric precursor materialwithin a mold (see, e.g., U.S. Pat. No. 5,512,131). In some instances,such polymeric substrate materials are preferred for their ease ofmanufacture, low cost, and disposability, as well as their generalinertness to most extreme reaction conditions. As with flow cell devicesfabricated from other materials, e.g., glass, flow cell devicesfabricated from these polymeric materials may include treated surfaces,e.g., derivatized or coated surfaces, to enhance their utility in themicrofluidic system, as will be discussed in more detail below.

The fluid channels and/or fluid chambers of the microfluidic devices aretypically fabricated into the upper surface of a first substrate asmicroscale channels (e.g., grooves, indentations, etc.) using the abovedescribed microfabrication techniques. The first substrate comprises atop side having a first planar surface and a bottom side. In themicrofluidic devices prepared in accordance with the methods describedherein, the plurality of fluid channels (e.g., grooves and/orindentations) are formed on the first planar surface. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surface (prior to bonding to a second substrate) have abottom and side walls, with the top remaining open. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surface (prior to bonding to a second substrate) have abottom and side walls and the top remaining closed. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surfaces (prior to bonding to a second substrate) have onlyside walls and no top or bottom surface (i.e., the fluid channels spanthe full thickness of the first substrate.

Fluid channels and chambers may be sealed by placing the first planarsurface of the first substrate in contact with, and bonding to, theplanar surface of a second substrate to form the channels and/orchambers (e.g., the interior portion) of the device at the interface ofthese two components. In some instances, after the first substrate isbonded to a second substrate, the structure may further be placed incontact with and bonded to a third substrate. In some instances, thethird substrate may be placed in contact with the side of the firstsubstrate that is not in contact with the second substrate. In someinstances, the first substrate is placed between the second substrateand the third substrate. In some instances, the second substrate and thethird substrate can cover and/or seal the grooves, indentations, orapertures formed on the first substrate to form the channels and/orchambers (e.g., the interior portion) of the device at the interface ofthese components.

The device can have openings that are oriented such that they are influid communication with at least one of the fluid channels and/or fluidchambers formed in the interior portion of the device, thereby formingfluid inlets and/or fluid outlets. In some instances, the openings areformed on the first substrate. In some instances, the openings areformed on the first and the second substrate. In some instances, theopenings are formed on the first, the second, and the third substrate.In some instances, the openings are positioned at the top side of thedevice. In some instances, the openings are positioned at the bottomside of the device. In some instances, the openings are positioned atthe first and/or the second ends of the device, and the channels runalong the direction from the first end to the second end.

Conditions under which substrates may be bonded together are generallywidely understood by those of skill in the art, and such bonding ofsubstrates is generally carried out by any of a variety of methods, thechoice of which may vary depending upon the nature of the substratematerials used. For example, thermal bonding of substrates may beapplied to a number of substrate materials including, e.g., glass orsilica-based substrates, as well as some polymer based-substrates. Suchthermal bonding techniques typically comprise mating the substratesurfaces that are to be bonded under conditions of elevated temperatureand, in some cases, application of external pressure. The precisetemperatures and pressures utilized will generally vary depending uponthe nature of the substrate materials used.

For example, for silica-based substrate materials, i.e., glass(borosilicate glass, Pyrex™, soda lime glass, etc.), fused-silica(quartz), and the like, thermal bonding of substrates is typicallycarried out at temperatures ranging from about 500° C. to about 1400°C., and preferably, from about 500° C. to about 1200° C. For example,soda lime glass is typically bonded at temperatures of around 550° C.,whereas borosilicate glass is typically thermally bonded at or near 800°C. Quartz substrates, on the other hand, are typically thermally bondedat temperatures at or near 1200° C. These bonding temperatures aretypically achieved by placing the substrates to be bonded into hightemperature annealing ovens.

Polymeric substrates that are thermally bonded, on the other hand, willtypically utilize lower temperatures and/or pressures than silica-basedsubstrates, in order to prevent excessive melting of the substratesand/or distortion, e.g., flattening of the interior portion of thedevice (i.e., the fluid channels or chambers). Generally, such elevatedtemperatures for bonding polymeric substrates will vary from about 80°C. to about 200° C., depending upon the polymeric material used, andwill preferably be between about 90° C. and about 150° C. Because of thesignificantly reduced temperatures required for bonding polymericsubstrates, such bonding may typically be carried out without the needfor the high temperature ovens used in the bonding of silica-basedsubstrates. This allows incorporation of a heat source within a singleintegrated bonding system, as described in greater detail below.

Adhesives may also be used to bond substrates together according towell-known methods, which typically comprise applying a layer ofadhesive between the substrates that are to be bonded and pressing themtogether until the adhesive sets. A variety of adhesives may be used inaccordance with these methods, including, e.g., UV curable adhesives,that are commercially available. Alternative methods may also be used tobond substrates together in accordance with the present invention,including e.g., acoustic or ultrasonic welding and/or solvent welding ofpolymeric parts.

Typically, a plurality of the described microfluidic chips or deviceswill be manufactured at the same time in parallel, e.g., using“wafer-scale” fabrication. For example, polymeric substrates may bestamped or molded in large separable sheets which can then be mated andbonded together. Individual devices or bonded substrates may then beseparated from the larger sheet by cutting or dicing. Similarly, forsilica-based substrates, individual devices can be fabricated fromlarger substrate wafers or plates, allowing higher throughput of themanufacturing process. Specifically, a plurality of fluid channelstructures can be fabricated on a first substrate wafer or plate, whichis then overlaid with and bonded to a second substrate wafer or plate,and optionally further overlaid with and bonded to a third substratewafer or plate. The individual devices are then segmented from thelarger substrates using known methods, such as sawing, scribing andbreaking, and the like.

As noted above, the top or second substrate is overlaid upon the bottomor first substrate to seal the various channels and chambers. Incarrying out the bonding process according to the methods of the presentdisclosure, the bonding of the first and second substrates may becarried out using vacuum and/or pressure to maintain the two substratesurfaces in optimal contact. In particular, the bottom substrate may bemaintained in optimal contact with the top substrate by, e.g., matingthe planar surface of the bottom substrate with the planar surface ofthe top substrate and applying a vacuum through holes that are disposedthrough the top substrate. Typically, application of a vacuum to holesin the top substrate is carried out by placing the top substrate on avacuum chuck, which typically comprises a mounting table or surface,having an integrated vacuum source. In the case of silica-basedsubstrates, the bonded substrates are subjected to elevated temperaturesin order to create an initial bond, so that the bonded substrates maythen be transferred to the annealing oven, without any shifting relativeto each other.

Alternate bonding systems for incorporation with the apparatus describedherein include, e.g., adhesive dispensing systems, for applying adhesivelayers between the two planar surfaces of the substrates. This may bedone by applying the adhesive layer prior to mating the substrates, orby placing an amount of the adhesive at one edge of the adjoiningsubstrates and allowing the wicking action of the two mated substratesto draw the adhesive across the space between the two substrates.

In certain instances, the overall bonding system can include automatablesystems for placing the top and bottom substrates on the mountingsurface and aligning them for subsequent bonding. Typically, suchsystems include translation systems for moving either the mountingsurface or one or more of the top and bottom substrates relative to eachother. For example, robotic systems may be used to lift, translate andplace each of the top and bottom substrates upon the mounting table, andwithin the alignment structures, in turn. Following the bonding process,such systems also can remove the finished product from the mountingsurface and transfer these mated substrates to a subsequent operation,e.g., a separation or dicing operation, an annealing oven forsilica-based substrates, etc., prior to placing additional substratesthereon for bonding.

In some instances, the manufacturing of the microfluidic chip includesthe layering or laminating of two or more layers of substrate, e.g.,patterned and non-patterned polymeric sheets, in order to produce thechip. For example, in microfluidic devices, the microfluidic features ofthe device are typically produced by laser irradiation, etching, orotherwise fabricating features into the surface of a first layer. Asecond layer is then laminated or bonded to the surface of the first toseal these features and provide the fluidic elements of the device,e.g., the fluid channels.

As noted above, in some instances one or more capillary flow celldevices or microfluidic chips may be mounted in a cartridge chassis toform a capillary flow cell cartridge or microfluidic cartridge. In someinstances, the capillary flow cell cartridge or microfluidic cartridgemay further comprise additional components that are integrated with thecartridge to provide enhanced performance for specific applications.Examples of additional components that may be integrated into thecartridge include, but are not limited to, adapters or connectors formaking fluidic connections to other components of the system, fluid flowcontrol components (e.g., miniature valves, miniature pumps, mixingmanifolds, etc.), temperature control components (e.g., resistiveheating elements, metal plates that serve as heat sources or sinks,piezoelectric (Peltier) devices for heating or cooling, temperaturesensors), or optical components (e.g., optical lenses, windows, filters,mirrors, prisms, fiber optics, and/or light-emitting diodes (LEDs) orother miniature light sources that may collectively be used tofacilitate spectroscopic measurements and/or imaging of one or morecapillary or fluid flow channels.

The fluidic adaptors, cartridge chassis, and other cartridge componentsmay be attached to the capillaries, capillary flow cell device(s),microfluidic chip(s) (or fluid channels within the chip) using any of avariety of techniques known to those of skill in the art including, butnot limited to, press fit, adhesive bonding, solvent bonding, laserwelding, etc., or any combination thereof. In some instances, theinlet(s) and/or outlet(s) of the microfluidic channels in themicrofluidic chip are apertures on the top surface of the chip, and thefluidic adaptors can be attached or coupled to the inlet(s) and/oroutlet(s) of the microfluidic channels within the chip. In someinstances, the cartridge may comprise additional adapters (i.e., inaddition to the fluidic adapters) that mate with the chip and/or fluidicadapters and help to position the chip within the cartridge. Theseadapters may be constructed using the same fabrication techniques andmaterials as those outlined above for the fluidic adapters.

The cartridge chassis (or “housing”) may be fabricated from metal and/orpolymer materials such as aluminum, anodized aluminum, polycarbonate(PC), acrylic (PMMA), or Ultem (PEI), while other materials are alsoconsistent with the present disclosure. A housing may be fabricatedusing CNC machining and/or molding techniques, and designed so that one,two, or more than two capillaries or microfluidic chips are constrainedby the chassis in a fixed orientation to create one or more independentflow channels. The capillaries or chips may be mounted in the chassisusing, e.g., a compression fit design, or by mating with compressibleadapters made of silicone or a fluoroelastomer. In some instances, twoor more components of the cartridge chassis (e.g., an upper half and alower half) are assembled using, e.g., screws, clips, clamps, or otherfasteners so that the two halves are separable. In some instances, twoor more components of the cartridge chassis are assembled using, e.g.,adhesives, solvent bonding, or laser welding so that the two or morecomponents are permanently attached.

Flow cell surface coatings: In some instances, one or more interiorsurfaces of the capillary lumens or microfluidic channels in thedisclosed flow cell devices (e.g., single- or multi-capillary flowcells, flow cell cartridges, microfluidic devices, or microfluidiccartridges) may be coated using any of a variety of surface modificationtechniques or polymer coatings described elsewhere herein. In someinstances, the coatings may be formulated to increase or maximize thenumber of available binding sites (e.g., tethered oligonucleotideadapter/primer sequences) on the one or more interior surfaces toincrease or maximize a foreground signal, e.g., a fluorescence signalarising from labeled nucleic acid molecules hybridized to tetheredoligonucleotide adapter/primer sequences. In some instances, thecoatings may be formulated to decrease or minimize nonspecific bindingof fluorophores and other small molecules, or labeled or unlabelednucleotides, proteins, enzymes, antibodies, oligonucleotides, or nucleicacid molecules (e.g., DNA, RNA, etc.), in order to decrease or minimizea background signal, e.g., background fluorescence arising from thenonspecific binding of labeled biomolecules or from autofluorescence ofa sample support structure. The combination of increased foregroundsignal and reduced background signal that may be achieved in someinstances through the use of the disclosed coatings may thus provideimproved signal-to-noise ratio (SNR) in spectroscopic measurements orimproved contrast-to-noise ratio (CNR) in imaging methods.

Fluidics systems and fluid flow control modules: in someimplementations, the disclosed imaging and/or analysis systems mayprovide fluid flow control capability for delivering samples or reagentsto the one or more flow cell devices or flow cell cartridges (e.g.,single capillary flow cell device or microfluidic channel flow celldevice) connected to the system. Reagents and buffers may be stored inbottles, reagent and buffer cartridges, or other suitable containersthat are connected to the flow cell inlets by means of tubing and valvemanifolds. The disclosed systems may also include processed sample andwaste reservoirs in the form of bottles, cartridges, or other suitablecontainers for collecting fluids downstream of the capillary flow celldevices or capillary flow cell cartridges. In some embodiments, thefluid flow (or “fluidics”) control module may provide programmableswitching of flow between different sources, e.g. sample or reagentreservoirs or bottles located in the instrument, and the inlet(s) to acentral region (e.g., a capillary flow cell or microfluidic device, or alarge fluid chamber such as a large fluid chamber within a microfluidicdevice). In some instances, the fluid flow control module may provideprogrammable switching of flow between outlet(s) from the central region(e.g., a capillary flow cell or microfluidic device) and differentcollection points, e.g., processed sample reservoirs, waste reservoirs,etc., connected to the system. In some instances, samples, reagents,and/or buffers may be stored within reservoirs that are integrated intothe flow cell cartridge or microfluidic cartridge itself. In someinstances, processed samples, spent reagents, and/or used buffers may bestored within reservoirs that are integrated into the flow cellcartridge or microfluidic device cartridge itself.

In some implementations, one or more fluid flow control modules may beconfigured to control the delivery of fluids to one or more capillaryflow cells, capillary flow cell cartridges, microfluidic devices,microfluidic cartridges, or any combination thereof. In some instances,the one or more fluidics controllers may be configured to controlvolumetric flow rates for one or more fluids or reagents, linear flowvelocities for one or more fluids or reagents, mixing ratios for one ormore fluids or reagents, or any combination thereof. Control of fluidflow through the disclosed systems will typically be performed usingpumps (or other fluid actuation mechanisms) and valves (e.g.,programmable pumps and valves). Examples of suitable pumps include, butare not limited to, syringe pumps, programmable syringe pumps,peristaltic pumps, diaphragm pumps, and the like. Examples of suitablevalves include, but are not limited to, check valves, electromechanicaltwo-way or three-way valves, pneumatic two-way and three-way valves, andthe like. In some instances, fluid flow through the system may becontrolled by means of applying positive pneumatic pressure to one ormore inlets of the reagent and buffer containers, or to inletsincorporated into flow cell cartridge(s) (e.g., capillary flow cell ormicrofluidic cartridges). In some embodiments, fluid flow through thesystem may be controlled by means of drawing a vacuum at one or moreoutlets of waste reservoir(s), or at one or more outlets incorporatedinto flow cell cartridge(s) (e.g., capillary flow cell or microfluidiccartridges).

In some instances, different modes of fluid flow control are utilized atdifferent points in an assay or analysis procedure, e.g. forward flow(relative to the inlet and outlet for a given capillary flow celldevice), reverse flow, oscillating or pulsatile flow, or combinationsthereof. In some applications, oscillating or pulsatile flow may beapplied, for example, during assay wash/rinse steps to facilitatecomplete and efficient exchange of fluids within the one or more flowcell devices or flow cell cartridges (e.g., capillary flow cell devicesor cartridges, and microfluidic devices or cartridges).

Similarly, in some cases different fluid flow rates may be utilized atdifferent locations within a flow cell device or at different points inthe assay or analysis process workflow, for example, in some instances,the volumetric flow rate may vary from −100 ml/sec to +100 ml/sec. Insome embodiment, the absolute value of the volumetric flow rate may beat least 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, atleast 1 ml/sec, at least 10 ml/sec, or at least 100 ml/sec. In someembodiments, the absolute value of the volumetric flow rate may be atmost 100 ml/sec, at most 10 ml/sec, at most 1 ml/sec, at most 0.1ml/sec, at most 0.01 ml/sec, or at most 0.001 ml/sec. The volumetricflow rate at a given location with the flow cell device or at a givenpoint in time may have any value within this range, e.g. a forward flowrate of 2.5 ml/sec, a reverse flow rate of −0.05 ml/sec, or a value of 0ml/sec (i.e., stopped flow).

In some implementations, the fluidics system may be designed to minimizethe consumption of key reagents (e.g., expensive reagents) required forperforming, e.g., genomic analysis applications. For example, in someimplementations the disclosed fluidics systems may comprise a firstreservoir housing a first reagent or solution, a second reservoirhousing a second reagent or solution, and a central region, e.g., acentral capillary flow cell or microfluidic device, where an outlet fromthe first reservoir and an outlet from the second reservoir arefluidically coupled to an inlet of the central capillary flow cell ormicrofluidic device through at least one valve such that the volume ofthe first reagent or solution flowing per unit time from the outlet ofthe first reservoir to the inlet of the central capillary flow cell ormicrofluidic device is less than the volume of the second reagent orsolution flowing per unit time from the outlet of the second reservoirto the inlet of the central region. In some implementations, the firstreservoir and second reservoir may be integrated into a capillary flowcell cartridge or microfluidic cartridge. In some instances, the atleast one valve may also be integrated into the capillary flow cellcartridge or microfluidic cartridge.

In some instances, the first reservoir is fluidically coupled to thecentral capillary flow cell or microfluidic device through a firstvalve, and the second reservoir is fluidically coupled to the centralcapillary flow cell or microfluidic device through a second valve. Insome instances, the first and/or second valves may be, e.g., a diaphragmvalve, pinch valve, gate valve, or other suitable valve. In someinstances, the first reservoir is positioned in close proximity to theinlet of the central capillary flow cell or microfluidic device toreduce dead volume for delivery of the first reagent solution. In someinstances, the first reservoir is placed in closer proximity to theinlet of the central capillary flow cell or microfluidic device than isthe second reservoir. In some instances, the first reservoir ispositioned in close proximity to the second valve so as to reduce thedead volume for delivery of the first reagent relative to that fordelivery of a plurality of “second” reagents (e.g., two, three, four,five, or six or more “second” reagents) from a plurality of “second”reservoirs (e.g., two, three, four, five, or six or more “second”reservoirs).

The first and second reservoirs described above may be used to house thesame or different reagents or solutions. In some instances, the firstreagent that is housed in the first reservoir is different from thesecond reagent that is housed in the second reservoir, and the secondreagent comprises at least one reagent that is used in common by aplurality of reactions occurring in the central a central capillary flowcell or microfluidic device. In some instances, e.g., in fluidicssystems configured for performing nucleic acid sequencing chemistrywithin the central capillary flow cell or microfluidic device, the firstreagent comprises at least one reagent selected from the groupconsisting of a polymerase, nucleotide, and a nucleotide analog. In someinstances, the second reagent comprises a low-cost reagent, e.g., asolvent.

In some instances, the interior volume of the central region, e.g., acentral capillary flow cell cartridge, or microfluidic device comprisingone or more fluid channels or fluid chambers, can be adjusted based onthe specific application to be performed, e.g., nucleic acid sequencing.In some embodiments, the central region comprises an interior volumesuitable for sequencing a eukaryotic genome. In some embodiments, thecentral region comprises an interior volume suitable for sequencing aprokaryotic genome. In some embodiments, the central region comprises aninterior volume suitable for sequencing a viral genome. In someembodiments, the central region comprises an interior volume suitablefor sequencing a transcriptome. For example, in some embodiments, theinterior volume of the central region may comprise a volume of less than0.05 μl, between 0.05 μl and 0.1 μl, between 0.05 μl and 0.2 μl, between0.05 μl and 0.5 μl, between 0.05 μl and 0.8 μl, between 0.05 μl and 1μl, between 0.05 μl and 1.2 between 0.05 μl and 1.5 μl, between 0.1 μland 1.5 μl, between 0.2 μl and 1.5 μl, between 0.5 μl and 1.5 μl,between 0.8 μl and 1.5 μl, between 1 μl and 1.5 μl, between 1.2 μl and1.5 or greater than 1.5 or a range defined by any two of the foregoing.In some embodiments, the interior volume of the central region maycomprise a volume of less than 0.5 μl, between 0.5 μl and 1 μl, between0.5 μl and 2 μl, between 0.5 μl and 5 μl, between 0.5 μl and 8 μl,between 0.5 μl and 10 μl, between 0.5 μl and 12 μl, between 0.5 μl and15 μl, between 1 μl and 15 μl, between 2 μl and 15 μl, between 5 μl and15 μl, between 8 μl and 15 μl, between 10 μl and 15 μl, between 12 μland 15 or greater than 15 or a range defined by any two of theforegoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 5 μl, between 5 μl and 10 μl,between 5 μl and 20 μl, between 5 μl and 500 μl, between 5 μl and 80 μl,between 5 μl and 100 μl, between 5 μl and 120 μl, between 5 μl and 150μl, between 10 μl and 150 μl, between 20 μl and 150 μl, between 50 μland 150 μl, between 80 μl and 150 μl, between 100 μl and 150 μl, between120 μl and 150 or greater than 150 or a range defined by any two of theforegoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 50 μl, between 50 μl and 100μl, between 50 μl and 200 μl, between 50 μl and 500 μl, between 50 μland 800 μl, between 50 μl and 1000 μl, between 50 μl and 1200 μl,between 50 μl and 1500 μl, between 100 μl and 1500 μl, between 200 μland 1500 μl, between 500 μl and 1500 μl, between 800 μl and 1500 μl,between 1000 μl and 1500 μl, between 1200 μl and 1500 μl, or greaterthan 1500 μl, or a range defined by any two of the foregoing. In someembodiments, the interior volume of the central region may comprise avolume of less than 500 μl, between 500 μl and 1000 μl, between 500 μland 2000 μl, between 500 μl and 5 ml, between 500 μl and 8 ml, between500 μl and 10 ml, between 500 μl and 12 ml, between 500 μl and 15 ml,between 1 ml and 15 ml, between 2 ml and 15 ml, between 5 ml and 15 ml,between 8 ml and 15 ml, between 10 ml and 15 ml, between 12 ml and 15ml, or greater than 15 ml, or a range defined by any two of theforegoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 5 ml, between 5 ml and 10 ml,between 5 ml and 20 ml, between 5 ml and 50 ml, between 5 ml and 80 ml,between 5 ml and 100 ml, between 5 ml and 120 ml, between 5 ml and 150ml, between 10 ml and 150 ml, between 20 ml and 150 ml, between 50 mland 150 ml, between 80 ml and 150 ml, between 100 ml and 150 ml, between120 ml and 150 ml, or greater than 150 ml, or a range defined by any twoof the foregoing. In some embodiments, the systems described hereincomprise an array or collection of flow cell devices or systemscomprising multiple discrete capillaries, microfluidic channels, fluidicchannels, chambers, or lumenal regions, wherein the combined interiorvolume is, comprises, or includes one or more of the values within arange disclosed herein.

In some instances, the ratio of volumetric flow rate for the delivery ofthe first reagent to the central capillary flow cell or microfluidicdevice to that for delivery of the second reagent to the centralcapillary flow cell or microfluidic device may be less than 1:20, lessthan 1:16, least than 1:12, less than 1:10, less than 1:8, less than1:6, or less than 1:2. In some instances, the ratio of volumetric flowrate for the delivery of the first reagent to the central capillary flowcell or microfluidic device to that for delivery of the second reagentto the central capillary flow cell or microfluidic device may have anyvalue with the range spanned by these values, e.g., less than 1:15.

As noted, the flow cell devices and/or fluidics systems disclosed hereinmay be configured to achieve a more efficient use of the reagents thanthat achieved by, e.g., other sequencing devices and systems,particularly for the costly reagents used in a variety of sequencingchemistry steps. In some instances, the first reagent comprises areagent that is more expensive than the second reagent. In someinstances, the first reagent comprises a reaction-specific reagent andthe second reagent comprises a nonspecific reagent common to allreactions performed in the central capillary flow cell or microfluidicdevice region, and wherein the reaction specific reagent is moreexpensive than the nonspecific reagent.

In some instances, utilization of the flow cell devices and/or fluidicsystems disclosed herein may convey advantages in terms of reducedconsumption of costly reagents. In some instances, for example,utilization of the flow cell devices and/or fluidic systems disclosedherein may results in at least a 5%, at least a 7.5%, at least a 10%, atleast a 12.5%, at least a 15%, at least a 17.5%, at least a 20%, atleast a 22.5%, at least a 25%, at least a 30%, at least a 35%, at leasta 40%, at least a 45%, or at least a 50% reduction in reagentconsumption compared to the reagent consumption encountered whenoperating, e.g., current commercially-available nucleic acid sequencingsystems.

FIG. 26 illustrates a non-limiting example of a simple fluidics systemcomprising a single capillary flow cell connected to various fluid flowcontrol components, where the single capillary is optically accessibleand compatible with mounting on a microscope stage or in a customimaging instrument for use in various imaging applications. A pluralityof reagent reservoirs is fluidically-coupled with the inlet end of thesingle capillary flow cell device, where the reagent flowing through thecapillary at any given point in time is controlled by means of aprogrammable rotary valve that allows the user to control the timing andduration of reagent flow. In this non-limiting example, fluid flow iscontrolled by means of a programmable syringe pump that provides precisecontrol and timing of volumetric fluid flow and fluid flow velocity.

Temperature control modules: In some implementations the disclosedsystems will include temperature control functionality for the purposeof facilitating the accuracy and reproducibility of assay or analysisresults. Examples of temperature control components that may beincorporated into the instrument system (or capillary flow cellcartridge) design include, but are not limited to, resistive heatingelements, infrared light sources, Peltier heating or cooling devices,heat sinks, thermistors, thermocouples, and the like. In some instances,the temperature control module (or “temperature controller”) may providefor a programmable temperature change at a specified, adjustable timeprior to performing specific assay or analysis steps. In some instances,the temperature controller may provide for programmable changes intemperature over specified time intervals. In some embodiments, thetemperature controller may further provide for cycling of temperaturesbetween two or more set temperatures with specified frequency and ramprates so that thermal cycling for amplification reactions may beperformed.

Fluid dispensing robotics: In some implementations, the disclosedsystems may comprise an automated, programmable fluid-dispensing (orliquid-dispensing) system for use in dispensing reagents or othersolutions into, e.g., microplates, capillary flow cell devices andcartridges, microfluidic devices and cartridges, etc. Suitableautomated, programmable fluid-dispensing systems are commerciallyavailable from a number of vendors, e.g. Beckman Coulter, Perkin Elmer,Tecan, Velocity 11, and many others. In a preferred aspect of thedisclosed systems, the fluid-dispensing system further comprises amultichannel dispense head, e.g. a 4 channel, 8 channel, 16 channel, 96channel, or 384 channel dispense head, for simultaneous delivery ofprogrammable volumes of liquid (e.g. ranging from about 1 microliter toseveral milliliters) to multiple wells or locations on a flow cellcartridge or microfluidic cartridge.

Cartridge- and/or microplate-handling (pick-and-place) robotics: In someimplementations, the disclosed system may comprise a cartridge- and/ormicroplate-handling robotic system for automated replacement andpositioning of microplates, capillary flow cell cartridges, ormicrofluidic device cartridges in relation to the optical imagingsystem, or for optionally moving microplates, capillary flow cellcartridges, or microfluidic device cartridges between the opticalimaging system and a fluid-dispensing system. Suitable automated,programmable microplate-handling robotic systems are commerciallyavailable from a number of vendors, including Beckman Coulter, PerkinElemer, Tecan, Velocity 11, and many others. In a preferred aspect ofthe disclosed systems, an automated microplate-handling robotic systemis configured to move collections of microwell plates comprising samplesand/or reagents to and from, e.g., refrigerated storage units.

Spectroscopy or imaging modules: As indicated above, in someimplementations the disclosed analysis systems will include opticalimaging capabilities and may also include other spectroscopicmeasurement capabilities. For example, the disclosed imaging modules maybe configured to operate in any of a variety of imaging modes known tothose of skill in the art including, but not limited to, bright-field,dark-field, fluorescence, luminescence, or phosphorescence imaging. Insome instances, the one or more capillary flow cells or microfluidicdevices of a fluidics sub-system comprise a window that allows at leasta section of one or more capillaries or one or more fluid channels ineach flow cell or microfluidic device to be illuminated and imaged.

In some embodiments, single wavelength excitation and emissionfluorescence imaging may be performed. In some embodiments, dualwavelength excitation and emission (or multi-wavelength excitation oremission) fluorescence imaging may be performed. In some instances, theimaging module is configured to acquire video images. The choice ofimaging mode may impact the design of the flow cells devices orcartridges in that all or a portion of the capillaries or cartridge willnecessarily need to be optically transparent over the spectral range ofinterest. In some instances, a plurality of capillaries within acapillary flow cell cartridge may be imaged in their entirety within asingle image. In some instances, only a single capillary or a subset ofcapillaries within a capillary flow cell cartridge, or portions thereof,may be imaged within a single image. In some instances, a series ofimages may be “tiled” to create a single high-resolution image of one,two, several, or the entire plurality of capillaries within a cartridge.In some instances, a plurality of fluid channels within a microfluidicchip may be imaged in their entirety within a single image. In someinstances, only a single fluid channel or a subset of fluid channelswithin a microfluidic chip, or portions thereof, may be imaged within asingle image. In some instances, a series of images may be “tiled” tocreate a single high-resolution image of one, two, several, or theentire plurality of fluid channels within a cartridge.

A spectroscopy or imaging module may comprise, e.g., a microscopeequipped with a CMOS of CCD camera. In some instances, the spectroscopyor imaging module may comprise, e.g., a custom instrument such as one ofthe imaging modules described herein that is configured to perform aspecific spectroscopic or imaging technique of interest. In general, thehardware associated with the spectroscopy or imaging module may includelight sources, detectors, and other optical components, as well asprocessors or computers.

Light sources: Any of a variety of light sources may be used to providethe imaging or excitation light, including but not limited to, tungstenlamps, tungsten-halogen lamps, arc lamps, lasers, light emitting diodes(LEDs), or laser diodes. In some instances, a combination of one or morelight sources, and additional optical components, e.g. lenses, filters,apertures, diaphragms, mirrors, and the like, may be configured as anillumination system (or sub-system).

Detectors: Any of a variety of image sensors may be used for imagingpurposes, including but not limited to, photodiode arrays,charge-coupled device (CCD) cameras, or complementarymetal-oxide-semiconductor (CMOS) image sensors. As used herein, “imagingsensors” may be one-dimensional (linear) or two-dimensional arraysensors. In many instances, a combination of one or more image sensors,and additional optical components, e.g. lenses, filters, apertures,diaphragms, mirrors, and the like, may be configured as an imagingsystem (or sub-system). In some instances, e.g., where spectroscopicmeasurements are performed by the system rather than imaging, suitabledetectors may include, but are not limited to, photodiodes, avalanchephotodiodes, and photomultipliers.

Other optical components: The hardware components of the spectroscopicmeasurement or imaging module may also include a variety of opticalcomponents for steering, shaping, filtering, or focusing light beamsthrough the system. Examples of suitable optical components include, butare not limited to, lenses, mirrors, prisms, apertures, diffractiongratings, colored glass filters, long-pass filters, short-pass filters,bandpass filters, narrowband interference filters, broadbandinterference filters, dichroic reflectors, optical fibers, opticalwaveguides, and the like. In some instances, as noted above, thespectroscopic measurement or imaging module may further comprise one ormore translation stages or other motion control mechanisms for thepurpose of moving capillary flow cell devices and cartridges relative tothe illumination and/or detection/imaging sub-systems, or vice versa.

Total internal reflection: In some instances, the optical module orsub-system may be designed to use all or a portion of an opticallytransparent wall of the capillaries or microfluidic channels in flowcell devices and cartridges as a waveguide for delivering excitationlight to the capillary or channel lumen(s) via total internalreflection. When incident excitation light strikes the surface of thecapillary or channel lumen at an angle with respect to a normal to thesurface that is larger than the critical angle (determined by therelative refractive indices of the capillary or channel wall materialand the aqueous buffer within the capillary or channel), total internalreflection occurs at the surface and the light propagates through thecapillary or channel wall along the length of the capillary or channel.Total internal reflection generates an evanescent wave at the lumensurface which penetrates the lumen interior for extremely shortdistances, and which may be used to selectively excite fluorophores atthe surface, e.g., labeled nucleotides that have been incorporated by apolymerase into a growing oligonucleotide through a solid-phase primerextension reaction.

Light-tight housings and environmental control chambers: In someimplementations, the disclosed systems may comprise a light-tighthousing to prevent stray ambient light from creating glare andobscuring, e.g., relatively faint fluorescence signals. In someimplementations, the disclosed systems may comprise an environmentalcontrol chamber that enables the system to operate under a tightlycontrolled temperature, humidity level, etc.

Processors and computers: In some instances, the disclosed systems maycomprise one or more processors or computers. The processor may be ahardware processor such as a central processing unit (CPU), a graphicprocessing unit (GPU), a general-purpose processing unit, or a computingplatform. The processor may be comprised of any of a variety of suitableintegrated circuits, microprocessors, logic devices, field-programmablegate arrays (FPGAs) and the like. In some instances, the processor maybe a single core or multi core processor, or a plurality of processorsmay be configured for parallel processing. Although the disclosure isdescribed with reference to a processor, other types of integratedcircuits and logic devices are also applicable. The processor may haveany suitable data operation capability. For example, the processor mayperform 512 bit, 256 bit, 128 bit, 64 bit, 32 bit, or 16 bit dataoperations.

The processor or CPU can execute a sequence of machine-readableinstructions, which can be embodied in a program or software. Theinstructions may be stored in a memory location. The instructions can bedirected to the CPU, which can subsequently program or otherwiseconfigure the CPU to implement, e.g., the system control methods of thepresent disclosure. Examples of operations performed by the CPU caninclude fetch, decode, execute, and write back.

Some processors may comprise a processing unit of a computer system. Thecomputer system may enable cloud-based data storage and/or computing. Insome instances, the computer system may be operatively coupled to acomputer network (“network”) with the aid of a communication interface.The network may be the internet, an intranet and/or extranet, anintranet and/or extranet that is in communication with the internet, ora local area network (LAN). The network in some cases is atelecommunication and/or data network. The network may include one ormore computer servers, which may enable distributed computing, such ascloud-based computing.

The computer system may also include computer memory or memory locations(e.g., random-access memory, read-only memory, flash memory), electronicstorage units (e.g., hard disk), communication interfaces (e.g., networkadapters) for communicating with one or more other systems, andperipheral devices, such as cache, other memory units, data storageunits and/or electronic display adapters. In some instances, thecommunication interface may allow the computer to be in communicationwith one or more additional devices. The computer may be able to receiveinput data from the coupled devices for analysis. Memory units, storageunits, communication interfaces, and peripheral devices may be incommunication with the processor or CPU through a communication bus(solid lines), such as may be incorporated into a motherboard. A memoryor storage unit may be a data storage unit (or data repository) forstoring data. The memory or storage units may store files, such asdrivers, libraries and saved programs. The memory or storage units maystore user data, e.g., user preferences and user programs.

The system control, image processing, and/or data analysis methods asdescribed herein can be implemented by way of machine-executable codestored in an electronic storage location of the computer system, suchas, for example, in the memory or electronic storage unit. Themachine-executable or machine-readable code can be provided in the formof software. During use, the code can be executed by the processor. Insome cases, the code can be retrieved from the storage unit and storedin memory for ready access by the processor. In some situations, theelectronic storage unit can be precluded, and machine-executableinstructions are stored in memory.

In some instances, the code may be pre-compiled and configured for usewith a machine having a processer adapted to execute the code. In someinstances, the code may be compiled during runtime. The code can besupplied in a programming language that can be selected to enable thecode to execute in a pre-compiled or as-compiled fashion.

Some aspects of the systems and methods provided herein can be embodiedin software. Various aspects of the technology may be thought of as“products” or “articles of manufacture” typically in the form of machine(or processor) executable code and/or associated data that is carried onor embodied in a type of machine-readable medium. Machine-executablecode can be stored on an electronic storage unit, such as memory (e.g.,read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory ofthe computers, processors or the like, or associated modules thereof,such as various semiconductor memories, tape drives, disk drives and thelike, which may provide non-transitory storage at any time for thesoftware programming. All or portions of the software may at times becommunicated through the Internet or various other telecommunicationnetworks. Such communications, for example, may enable loading of thesoftware from one computer or processor into another, for example, froma management server or host computer into the computer platform of anapplication server. Thus, another type of media that may bear thesoftware elements includes optical, electrical and electromagneticwaves, such as used across physical interfaces between local devices,through wired and optical landline networks and over various air-links.The physical elements that carry such waves, such as wired or wirelesslinks, optical links or the like, also may be considered as mediabearing the software. As used herein, unless restricted tonon-transitory, tangible “storage” media, terms such as computer ormachine “readable medium” refer to any medium that participates inproviding instructions to a processor for execution.

In some instances, the system control, image processing, and/or dataanalysis methods of the present disclosure may be implemented by way ofone or more algorithms. An algorithm may be implemented by way ofsoftware upon execution by the central processing unit.

System control software: In some instances, the system may comprise acomputer (or processor) and a computer-readable medium that includescode for providing a user interface as well as manual, semi-automated,or fully-automated control of all system functions, e.g., control of thefluid flow control module(s), the temperature control module(s), and/orthe spectroscopy or imaging module(s), as well as other data analysisand display options. The system computer or processor may be anintegrated component of the system (e.g. a microprocessor or motherboard embedded within the instrument) or may be a stand-alone module,for example, a main frame computer, a personal computer, or a laptopcomputer. Examples of fluid flow control functions provided by thesystem control software include, but are not limited to, volumetricfluid flow rates, fluid flow velocities, the timing and duration forsample and reagent addition, buffer addition, and rinse steps. Examplesof temperature control functions provided by the system control softwareinclude, but are not limited to, specifying temperature set point(s) andcontrol of the timing, duration, and ramp rates for temperature changes.Examples of spectroscopic measurement or imaging control functionsprovided by the system control software include, but are not limited to,autofocus capability, control of illumination or excitation lightexposure times and intensities, control of image acquisition rate,exposure time, and data storage options.

Image processing software: In some instances, the system may furthercomprise a computer (or processor) and computer-readable medium thatincludes code for providing image processing and analysis capability.Examples of image processing and analysis capability that may beprovided by the software include, but are not limited to, manual,semi-automated, or fully-automated image exposure adjustment (e.g. whitebalance, contrast adjustment, signal-averaging and other noise reductioncapability, etc.), automated edge detection and object identification(e.g., for identifying clonally-amplified clusters offluorescently-labeled oligonucleotides on the lumen surface of capillaryflow cell devices), automated statistical analysis (e.g., fordetermining the number of clonally-amplified clusters ofoligonucleotides identified per unit area of the capillary lumensurface, or for automated nucleotide base-calling in nucleic acidsequencing applications), and manual measurement capabilities (e.g. formeasuring distances between clusters or other objects, etc.).Optionally, instrument control and image processing/analysis softwaremay be written as separate software modules. In some embodiments,instrument control and image processing/analysis software may beincorporated into an integrated package.

Any of a variety of image processing methods known to those of skill inthe art may be used for image processing/pre-processing. Examplesinclude, but are not limited to, Canny edge detection methods,Canny-Deriche edge detection methods, first-order gradient edgedetection methods (e.g., the Sobel operator), second order differentialedge detection methods, phase congruency (phase coherence) edgedetection methods, other image segmentation algorithms (e.g., intensitythresholding, intensity clustering methods, intensity histogram-basedmethods, etc.), feature and pattern recognition algorithms (e.g., thegeneralized Hough transform for detecting arbitrary shapes, the circularHough transform, etc.), and mathematical analysis algorithms (e.g.,Fourier transform, fast Fourier transform, wavelet analysis,auto-correlation, etc.), or any combination thereof.

Nucleic acid sequencing systems & applications: Nucleic acid sequencing,e.g., cellularly-addressable nucleic acid sequencing, provides onenon-limiting example of an application for the disclosed flow celldevices (e.g., capillary flow cell devices or cartridges, andmicrofluidic devices and cartridges) and imaging systems. Theimprovements in flow cell device design disclosed herein, e.g.,comprising hydrophilic coated surfaces that maximize foreground signalsfor, e.g., fluorescently-labeled nucleic acid clusters disposed thereon,while minimizing background signal may give rise to improvements in CNRfor images used for base-calling purposes, in combination withimprovements in optical imaging system design for fast dual-surface flowcell imaging (comprising simultaneous or near-simultaneous imaging ofthe interior flow cell surfaces) achieved through improved objectivelens and/or tube lens designs that provide for larger depth of field andlarger fields-of-view, and reduced reagent consumption (achieved throughimproved flow cell design) may give rise to dramatic improvements inbase-calling accuracy, shortened imaging cycle times, shortened overallsequencing reaction cycle times, and higher throughput nucleic acidsequencing at reduced cost per base.

In some instances, the disclosed hydrophilic, polymer coated flow celldevices used in combination with the optical imaging systems disclosedherein may confer one or more of the following additional advantages fora nucleic acid sequencing system: (i) decreased fluidic wash times (dueto reduced non-specific binding, and thus faster sequencing cycletimes), (ii) decreased imaging times (and thus faster turnaround timesfor assay readout and sequencing cycles), (iii) decreased overall workflow time requirements (due to decreased cycle times), (iv) decreaseddetection instrumentation costs (due to the improvements in CNR), (v)improved readout (base-calling) accuracy (due to improvements in CNR),(vi) improved reagent stability and decreased reagent usage requirements(and thus reduced reagents costs), and (vii) fewer run-time failures dueto nucleic acid amplification failures.

Flow cell devices configured for sequencing: In some instances, one ormore flow cell devices according to the present disclosure may beconfigured for nucleic acid sequencing applications, e.g., wherein twoor more interior flow cell device surfaces comprise hydrophilic polymercoatings, as disclosed elsewhere herein, that further comprise one ormore capture oligonucleotides, e.g., adapter/primer oligonucleotides, orany other oligonucleotides as disclosed elsewhere herein. In someinstances, the hydrophilic, polymer-coated surfaces of the disclosedflow cell devices may comprise a plurality of oligonucleotides tetheredthereto that have been selected for use in sequencing a eukaryoticgenome. In some instances, the hydrophilic, polymer-coated surfaces ofthe disclosed flow cell devices may comprise a plurality ofoligonucleotides tethered thereto that have been selected for use insequencing a prokaryotic genome or portion thereof. In some instances,the hydrophilic, polymer-coated surfaces of the disclosed flow celldevices may comprise a plurality of oligonucleotides tethered theretothat have been selected for use in sequencing a viral genome or portionthereof. In some instances, the hydrophilic, polymer-coated surfaces ofthe disclosed flow cell devices may comprise a plurality ofoligonucleotides tethered thereto that have been selected for use insequencing a transcriptome.

In some instances, a flow cell device of the present disclosure maycomprise a first surface in an orientation generally facing the interiorof the flow channel, a second surface in an orientation generally facingthe interior of the flow channel and further generally facing orparallel to the first surface, a third surface generally facing theinterior of a second flow channel, and a fourth surface, generallyfacing the interior of the second flow channel and generally opposed toor parallel to the third surface; wherein said second and third surfacesmay be located on or attached to opposite sides of a generally planarsubstrate which may be a reflective, transparent, or translucentsubstrate. In some instances, an imaging surface or imaging surfaceswithin a flow cell may be located within the center of a flow cell orwithin or as part of a division between two subunits or subdivisions ofa flow cell, wherein said flow cell may comprise a top surface and abottom surface, one or both of which may be transparent to suchdetection mode as may be utilized; and wherein a surface comprisingoligonucleotides adapters/primers tethered to one or more polymercoatings may be placed or interposed within the lumen of the flow cell.In some instances, the top and/or bottom surfaces do not includeattached oligonucleotide adapters/primers. In some instances, said topand/or bottom surfaces do comprise attached oligonucleotideadapters/primers. In some instances, either said top or said bottomsurface may comprise attached oligonucleotide adapters/primers. Asurface or surfaces placed or interposed within the lumen of a flow cellmay be located on or attached to one side, to an opposite side, or toboth sides of a generally planar substrate which may be a reflective,transparent, or translucent substrate.

Fluorescence imaging of hydrophilic, polymer-coated flow cell devicesurfaces: The disclosed hydrophilic, polymer-coated flow cell devicescomprising, e.g., clonal clusters of labeled target nucleic acidmolecules disposed thereon may be used in any of a variety of nucleicacid analysis applications, e.g., nucleic acid base discrimination,nucleic acid base classification, nucleic acid base calling, nucleicacid detection applications, nucleic acid sequencing applications, andnucleic acid-based (genetic and genomic) diagnostic applications. Inmany of these applications, fluorescence imaging techniques may be usedto monitor hybridization, amplification, and/or sequencing reactionsperformed on the low-binding supports. Fluorescence imaging may beperformed using any of the optical imaging modules disclosed herein, aswell as a variety of fluorophores, fluorescence imaging techniques, andother fluorescence imaging instruments known to those of skill in theart.

Nucleic acid sequencing system performance: In some instances, thedisclosed nucleic acid sequencing systems, comprising one or more of thedisclosed flow cell devices used in combination with one or more of thedisclosed optical imaging systems, and optionally utilizing one of theemerging sequencing biochemistries such as the “sequencing-by-nucleotidebinding” approach described in U.S. Pat. No. 10,655,176 B2, and the“sequencing-by-avidity” approach described in U.S. Pat. No. 10,768,173B2 instead of more conventional sequencing-by-nucleotide incorporationapproaches, may provide improved nucleic acid sequencing performance interms of, e.g., reduced sample input requirements, reduced imageacquisition cycle time, reduced sequencing reaction cycle time, reducedsequencing run time, improved base-calling accuracy, reduced reagentconsumption and cost, higher sequencing throughput, and reducedsequencing cost.

Nucleic acid sample input (pM): In some instances, the sample inputrequirements for the disclosed system may be significantly reduced dueto the improved hybridization and amplification efficiencies that may beattained, and the high CNR images that may be acquired for base-calling,using the disclosed hydrophilic, polymer coated flow cell devices andimaging systems. In some instances, the nucleic acid sample inputrequirement for the disclosed systems may range from about 1 pM to about10,000 pM. In some instances, the nucleic acid sample input requirementmay be at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, atleast 20 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least500 pM, at least 1,000 pM, at least 2,000 pM, at least 5,000 pM, atleast 10,000 pM. In some instances, the nucleic acid sample inputrequirement for the disclosed systems may be at most 10,000 pM, at most5,000 pM, at most 2,000 pM, at most 1,000 pM, at most 500 pM, at most200 pM, at most 100 pM, at most 50 pM, at most 20 pM, at most 10 pM, atmost 5 pM, at most 2 pM, or at most 1 pM. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe nucleic acid sample input requirement for the disclosed systems mayrange from about 5 pM to about 500 pM. Those of skill in the art willrecognize that the nucleic acid sample input requirement may have anyvalue within this range, e.g., about 132 pM. In one exemplary instance,a nucleic acid sample input of about 100 pM is sufficient to generatesignals for reliable base-calling.

Nucleic acid sample input (nanograms): In some instances, the nucleicacid sample input requirement for the disclosed systems may range fromabout 0.05 nanograms to about 1,000 nanograms. In some instances, thenucleic acid sample input requirement may be at least 0.05 nanograms, atleast 0.1 nanograms, at least 0.2 nanograms, at least 0.4 nanograms, atleast 0.6 nanograms, at least 0.8 nanograms, at least 1.0 nanograms, atleast 2 nanograms, at least 4 nanograms, at least 6 nanograms, at least8 nanograms, at least 10 nanograms, at least 20 nanograms, at least 40nanograms, at least 60 nanograms, at least 80 nanograms, at least 100nanograms, at least 200 nanograms, at least 400 nanograms, at least 600nanograms, at least 800 nanograms, or at least 1,000 nanograms. In someinstances, the nucleic acid sample input requirement may be at most1,000 nanograms, at most 800 nanograms, at most 600 nanograms, at most400 nanograms, at most 200 nanograms, at most 100 nanograms, at most 80nanograms, at most 60 nanograms, at most 40 nanograms, at most 20nanograms, at most 10 nanograms, at most 8 nanograms, at most 6nanograms, at most 4 nanograms, at most 2 nanograms, at most 1nanograms, at most 0.8 nanograms, at most 0.6 nanograms, at most 0.4nanograms, at most 0.2 nanograms, at most 0.1 nanograms, or at most 0.05nanograms. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the nucleic acid sample input requirementfor the disclosed systems may range from about 0.6 nanograms to about400 nanograms. Those of skill in the art will recognize that the nucleicacid sample input requirement may have any value within this range,e.g., about 2.65 nanograms.

# FOV images required to tile flow cell: In some instances, thefield-of-view (FOV) of the disclosed optical imaging module issufficiently large that a multi-channel (or multi-lane) flow cell (i.e.,the fluid channel portions thereof) of the present disclosure may beimaged by tiling from about 10 FOV images (or “frames”) to about 1,000FOV images (or “frames”). In some instances, an image of the entiremulti-channel flow cell may require tiling at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 300, at least 350, at least 400, at least 450, at least 500, atleast 550, at least 600, at least 650, at least 700, at least 750, atleast 800, at least 850, at least 900, at least 950, or at least 1,000FOV images (or “frames”). In some instances, an image of the entiremulti-channel flow cell may require tiling at most 1,000, at most 950,at most 900, at most 850, at most 800, at most 750, at most 700, at most650, at most 600, at most 550, at most 500, at most 450, at most 400, atmost 350, at most 300, at most 250, at most 200, at most 150, at most100, at most 90, at most 80, at most 80, at most 70, at most 60, at most50, at most 40, at most 30, at most 20, or at most 10 FOV images (or“frames”). Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances an image of the entire multi-channel flowcell may require tiling from about 30 to about 100 FOV images. Those ofskill in the art will recognize that in some instances the number ofrequired FOV images may have any value within this range, e.g., about 54FOV images.

Imaging cycle time: In some instances, the combination of large FOV,image sensor response sensitivity, and/or fast FOV translation timesenables shortened imaging cycle times (i.e., the time required toacquire a sufficient number of FOV images to tile the entiremultichannel flow cell (or the fluid channel portions thereof). In someinstances, the imaging cycle time may range from about 10 seconds toabout 10 minutes. In some instances, the imaging cycle time may be atleast 10 seconds at least 20 seconds, at least 30 seconds, at least 40seconds, at least 50 seconds, at least 1 minute, at least 2 minutes, atleast 3 minutes, at least 4 minutes, at least 5 minutes, at least 6minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, orat least 10 minutes. In some instances, the imaging cycle time may be atmost 10 minutes, at most 9 minutes, at most 8 minutes, at most 7minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, atmost 3 minutes, at most 2 minutes, at most 1 minute, at most 50 second,at most 40 second, at most 30 seconds, at most 20 seconds, or at most 10seconds. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the imaging cycle time may range fromabout 20 seconds to about 1 minute. Those of skill in the art willrecognize that in some instances the imaging cycle time may have anyvalue within this range, e.g., about 57 seconds.

Sequencing cycle time: In some instances, shortened sequencing reactionsteps, e.g., due to reduced wash time requirements for the disclosedhydrophilic, polymer-coated flow cells, may result in shortened overallsequencing cycle times. In some instances, the sequencing cycle timesfor the disclosed systems may range from about 1 minute to about 60minutes. In some instances, the sequencing cycle time may be at least 1minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, atleast 5 minutes, at least 6 minutes, at least 7 minutes, at least 8minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes,at least 20 minutes, at least 25 minutes, at least 30 minutes, at least35 minutes, at least 40 minutes, at least 45 minutes, at least 50minutes, at least 55 minutes, or at least 60 minutes. In some instances,the sequencing reaction cycle time may be at most 60 minutes, at most 55minutes, at most 50 minutes, at most 45 minutes, at most 40 minutes, atmost 35 minutes, at most 30 minutes, at most 25 minutes, at most 20minutes, at most 15 minutes, at most 10 minutes, at most 9 minutes, atmost 8 minutes, at most 7 minutes, at most 6 minutes, at most 5 minutes,at most 4 minutes, at most 3 minutes, at most 2 minutes, or at most 1minutes. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the sequencing cycle time may range fromabout 2 minutes to about 15 minutes. Those of skill in the art willrecognize that in some instances the sequencing cycle time may have anyvalue within this range, e.g., about 1 minute, 12 seconds.

Sequencing read length: In some instances, the enhanced CNR images thatmay be achieved using the disclosed hydrophilic, polymer-coated flowcell devices in combination with the disclosed imaging systems, and insome cases, the use of milder sequencing biochemistries, may enablelonger sequencing read lengths for the disclosed systems. In someinstances, the maximum (single read) read length may range from about 50bp to about 500 bp. In some instances, the maximum (single read) readlength may be at least 50 bp, at least 100 bp, at least 150 bp, at least200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400bp, at least 450 bp, or at least 500 bp. In some instances, the maximum(single read) read length is at most 500 bp, at most 450 bp, at most 400bp, at most 350 bp, at most 300 bp, at most 250 bp, at most 200 bp, atmost 150 bp, at most 100 bp, or at most 50 bp. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe maximum (single read) read length may range from about 100 bp toabout 450 bp. Those of skill in the art will recognize that in someinstances the maximum (single read) read length may have any valuewithin this range, e.g., about 380 bp.

Sequencing run time: In some instances, the sequencing run time for thedisclosed nucleic acid sequencing systems may range from about 8 hoursto about 20 hours. In some instances, the sequencing run time is atleast 8 hours, at least 9 hours, at least 10 hours, at least 12 hours,at least 14 hours, at least 16 hours, at least 18 hours, or at least 20hours. In some instances, the sequencing run time is at most 20 hours,at most 18 hours, at most 16 hours, at most 14 hours, at most 12 hours,at most 10 hours, at most 9 hours, or at most 8 hours. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the sequencing run time may range from about 10 hours to about16 hours. Those of skill in the art will recognize that in someinstances the sequencing run time may have any value within this range,e.g., about 7 hours, 35 minutes.

Average base-calling accuracy: In some instances, the disclosed nucleicacid sequencing systems may provide an average base-calling accuracy ofat least 80%, at least 85%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9% correct over the course of a sequencing run. In someinstances, the disclosed nucleic acid sequencing systems may provide anaverage base-calling accuracy of at least 80%, at least 85%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99%, at least 99.5%, at least 99.8%, or at least 99.9% correct per every1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or100,000 bases called.

Average Q-score: In some instances, the disclosed nucleic acidsequencing systems may provide a more accurate base readout. In someinstances, for example, the disclosed nucleic acid sequencing systemsmay provide an average Q-score for base-calling accuracy over asequencing run that ranges from about 20 to about 50. In some instances,the average Q-score may be at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50. Those of skill inthe art will recognize that the average Q-score may have any valuewithin this range, e.g., about 32.

Q-score vs. % nucleotides identified: In some instances, the disclosednucleic acid sequencing systems may provide a Q-score of greater than 30for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some instances, thedisclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 35 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 40 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 45 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed compositions and methods for nucleic acid sequencing mayprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

Reagent consumption: In some instances, the disclosed nucleic acidsequencing systems may have lower reagent consumption rates and costsdue to, e.g., the use of the disclosed flow cell devices and fluidicsystems that minimize fluid channel volumes and dead volumes. In someinstances, the disclosed nucleic acid sequencing systems may thusrequire an average of at least 5% less, at least 10% less, at least 15%less, at least 20% less, at least 25% less, at least 30% less, at least35% less, at least 40% less, at least 45% less, or at least 50% lessreagent by volume per Gbase sequenced that that consumed by an IlluminaMiSeq sequencer.

Sequencing throughput: In some instances, the disclosed nucleic acidsequencing systems may provide a sequencing throughput ranging fromabout 50 Gbase/run to about 200 Gbase/run. In some instances, thesequencing throughput may be at least 50 Gbase/run, at least 75Gbase/run, at least 100 Gbase/run, at least 125 Gbase/run, at least 150Gbase/run, at least 175 Gbase/run, or at least 200 Gbase/run. In someinstances, the sequencing throughput may be at most 200 Gbase/run, atmost 175 Gbase/run, at most 150 Gbase/run, at most 125 Gbase/run, atmost 100 Gbase/run, at most 75 Gbase/run, or at most 50 Gbase/run. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the sequencing throughput may range fromabout 75 Gbase/run to about 150 Gbase/run. Those of skill in the artwill recognize that in some instances the sequencing throughput may haveany value within this range, e.g., about 119 Gbase/run.

Sequencing cost: In some instances, the disclosed nucleic acidsequencing systems may provide nucleic acid sequencing at a cost rangingfrom about $5 per Gbase to about $30 per Gbase. In some instances, thesequencing cost may be at least $5 per Gbase, at least $10 per Gbase, atleast $15 per Gbase, at least $20 per Gbase, at least $25 per Gbase, orat least $30 per Gbase. In some instances, the sequencing cost may be atmost $30 per Gbase, at most $25 per Gbase, at most $20 per Gbase, atmost $15 per Gbase, at most $10 per Gbase, or at most $30 per Gbase. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the sequencing cost may range from about $10per Gbase to about $15 per Gbase. Those of skill in the art willrecognize that in some instances the sequencing cost may have any valuewithin this range, e.g., about $7.25 per Gbase.

Enablement of optical systems is further provided in U.S. patentapplication Ser. No. 16/363,842, hybridization methods as disclosed inU.S. patent application Ser. No. 17/016,349, U.S. patent applicationSer. No. 17/016,350, and U.S. patent application Ser. No. 17/016,353,the contents of which are hereby expressly incorporated by reference forall purposes.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

Generally, terminologies pertaining to techniques of molecular biology,nucleic acid chemistry, protein chemistry, genetics, microbiology,transgenic cell production, and hybridization described herein are thosewell-known and commonly used in the art. Techniques and proceduresdescribed herein are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout theinstant specification. For example, see Sambrook et al., MolecularCloning: A Laboratory Manual (Third ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques described herein are those well-known and commonly usedin the art.

Throughout this application, various embodiments may be presented in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosure. Accordingly,the description of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a sample” includes a plurality ofsamples, including mixtures thereof.

The term “and/or” used herein is to be taken mean specific disclosure ofeach of the specified features or components with or without the other.For example, the term “and/or” as used in a phrase such as “A and/or B”herein is intended to include: “A and B”; “A or B”; “A” (A alone); and“B” (B alone). In a similar manner, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “Bor C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone);and “C” (C alone).

As used herein and in the appended claims, terms “comprising”,“including”, “having” and “containing”, and their grammatical variants,as used herein are intended to be non-limiting so that one item ormultiple items in a list do not exclude other items that can besubstituted or added to the listed items. It is understood that whereveraspects are described herein with the language “comprising,” otherwiseanalogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

The terms “determining,” “measuring,” “evaluating,” “assessing,”“assaying,” and “analyzing” are often used interchangeably herein torefer to forms of measurement. The terms include determining if anelement is present or not (for example, detection). These terms caninclude quantitative, qualitative or quantitative and qualitativedeterminations. Assessing can be relative or absolute. “Detecting thepresence of” can include determining the amount of something present inaddition to determining whether it is present or absent depending on thecontext.

The terms “subject,” “individual,” or “patient” are often usedinterchangeably herein. A “subject” can be a biological entitycontaining expressed genetic materials. The biological entity can be aplant, animal, or microorganism, including, for example, bacteria,viruses, fungi, and protozoa. The subject can be tissues, cells andtheir progeny of a biological entity obtained in vivo or cultured invitro. The subject can be a mammal. The mammal can be a human. Thesubject may be diagnosed or suspected of being at high risk for adisease. In some cases, the subject is not necessarily diagnosed orsuspected of being at high risk for the disease.

The term “in vivo” is used to describe an event that takes place in asubject's body.

The term “ex vivo” is used to describe an event that takes place outsideof a subject's body. An ex vivo assay is not performed on a subject.Rather, it is performed upon a sample separate from a subject. Anexample of an ex vivo assay performed on a sample is an “in vitro”assay.

The term “in vitro” is used to describe an event that takes placescontained in a container for holding laboratory reagent such that it isseparated from the biological source from which the material isobtained. In vitro assays can encompass cell-based assays in whichliving or dead cells are employed. In vitro assays can also encompass acell-free assay in which no intact cells are employed.

As used herein, the terms “about” and “approximately” refer to a valueor composition that is within an acceptable error range for theparticular value or composition as determined by one of ordinary skillin the art, which will depend in part on how the value or composition ismeasured or determined, e.g., the limitations of the measurement system.For example, “about” or “approximately” can mean within one or more thanone standard deviation per the practice in the art. Alternatively,“about” or “approximately” can mean a range of up to 10% (i.e., ±10%) ormore depending on the limitations of the measurement system. Forexample, about 5 mg can include any number between 4.5 mg and 5.5 mg.Furthermore, particularly with respect to biological systems orprocesses, the terms can mean up to an order of magnitude or up to5-fold of a value. When particular values or compositions are providedin the instant disclosure, unless otherwise stated, the meaning of“about” or “approximately” should be assumed to be within an acceptableerror range for that particular value or composition. Also, where rangesand/or subranges of values are provided, the ranges and/or subranges caninclude the endpoints of the ranges and/or subranges.

The term “polymerase” and its variants, as used herein, comprises anenzyme comprising a domain that binds a nucleotide (or nucleoside) wherethe polymerase can form a complex having a template nucleic acid and acomplementary nucleotide. The polymerase can have one or more activitiesincluding, but not limited to, base analog detection activities, DNApolymerization activity, reverse transcriptase activity, DNA binding,strand displacement activity, and nucleotide binding and recognition. Apolymerase can be any enzyme that can catalyze polymerization ofnucleotides (including analogs thereof) into a nucleic acid strand.Typically but not necessarily such nucleotide polymerization can occurin a template-dependent fashion. Typically, a polymerase comprises oneor more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, a polymeraseincludes other enzymatic activities, such as for example, 3′ to 5′exonuclease activity or 5′ to 3′ exonuclease activity. In someembodiments, a polymerase has strand displacing activity. A polymerasecan include without limitation naturally occurring polymerases and anysubunits and truncations thereof, mutant polymerases, variantpolymerases, recombinant, fusion or otherwise engineered polymerases,chemically modified polymerases, synthetic molecules or assemblies, andany analogs, derivatives or fragments thereof that retain the ability tocatalyze nucleotide polymerization (e.g., catalytically activefragment). The polymerase includes catalytically inactive polymerases,catalytically active polymerases, reverse transcriptases, and otherenzymes comprising a nucleotide binding domain. In some embodiments, apolymerase can be isolated from a cell, or generated using recombinantDNA technology or chemical synthesis methods. In some embodiments, apolymerase can be expressed in prokaryote, eukaryote, viral, or phageorganisms. In some embodiments, a polymerase can be post-translationallymodified proteins or fragments thereof. A polymerase can be derived froma prokaryote, eukaryote, virus or phage. A polymerase comprisesDNA-directed DNA polymerase and RNA-directed DNA polymerase.

As used herein, the term “strand displacing” refers to the ability of apolymerase to locally separate strands of double-stranded nucleic acidsand synthesize a new strand in a template-based manner. Stranddisplacing polymerases displace a complementary strand from a templatestrand and catalyze new strand synthesis. Strand displacing polymerasesinclude mesophilic and thermophilic polymerases. Strand displacingpolymerases include wild type enzymes, and variants includingexonuclease minus mutants, mutant versions, chimeric enzymes andtruncated enzymes. Examples of strand displacing polymerases includephi29 DNA polymerase, large fragment of Bst DNA polymerase, largefragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), Klenowfragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” andother related terms used herein are used interchangeably and refer topolymers of nucleotides and are not limited to any particular length.Nucleic acids include recombinant and chemically-synthesized forms.Nucleic acids can be isolated. Nucleic acids include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs (e.g., peptide nucleicacids (PNA) and non-naturally occurring nucleotide analogs), andchimeric forms containing DNA and RNA. Nucleic acids can besingle-stranded or double-stranded. Nucleic acids comprise polymers ofnucleotides, where the nucleotides include natural or non-natural basesand/or sugars. Nucleic acids comprise naturally-occurringinternucleosidic linkages, for example phosphdiester linkages. Nucleicacids can lack a phosphate group. Nucleic acids comprise non-naturalinternucleoside linkages, including phosphorothioate, phosphorothiolate,or peptide nucleic acid (PNA) linkages. In some embodiments, nucleicacids comprise a one type of polynucleotides or a mixture of two or moredifferent types of polynucleotides.

The term “primer” and related terms used herein refers to anoligonucleotide that is capable of hybridizing with a DNA and/or RNApolynucleotide template to form a duplex molecule. Primers comprisenatural nucleotides and/or nucleotide analogs. Primers can berecombinant nucleic acid molecules. Primers may have any length, buttypically range from 4-50 nucleotides. A typical primer comprises a 5′end and 3′ end. The 3′ end of the primer can include a 3′ OH moietywhich serves as a nucleotide polymerization initiation site in apolymerase-catalyzed primer extension reaction. Alternatively, the 3′end of the primer can lack a 3′ OH moiety, or can include a terminal 3′blocking group that inhibits nucleotide polymerization in apolymerase-catalyzed reaction. Any one nucleotide, or more than onenucleotide, along the length of the primer can be labeled with adetectable reporter moiety. A primer can be in solution (e.g., a solubleprimer) or can be immobilized to a support (e.g., a capture primer).

The nucleic acids of interest can be extracted from cells or biologicalsample s using any of a number of techniques known to those of skill inthe art. For example, a typical DNA extraction procedure comprises (i)collection of the cell sample or tissue sample from which DNA is to beextracted, (ii) disruption of cell membranes (i.e., cell lysis) torelease DNA and other cytoplasmic components, (iii) treatment of thelysed sample with a concentrated salt solution to precipitate proteins,lipids, and RNA, followed by centrifugation to separate out theprecipitated proteins, lipids, and RNA, and (iv) purification of DNAfrom the supernatant to remove detergents, proteins, salts, or otherreagents used during the cell membrane lysis. A variety of suitablecommercial nucleic acid extraction and purification kits are consistentwith the disclosure herein. Examples include, but are not limited to,the QIAamp kits (for isolation of genomic DNA from human samples) andDNAeasy kits (for isolation of genomic DNA from animal or plant samples)from Qiagen (Germantown, Md.), or the Maxwell® and ReliaPrep™ series ofkits from Promega (Madison, Wis.).

The term “template nucleic acid”, “template polynucleotide”, “targetnucleic acid” “target polynucleotide”, “template strand” and othervariations refer to a nucleic acid strand that serves as the basisnucleic acid molecule for any of the analysis methods describe herein(e.g., amplifying and/or sequencing). The template nucleic acid can besingle-stranded or double-stranded, or the template nucleic acid canhave single-stranded or double-stranded portions. The template nucleicacid can be obtained from a naturally-occurring source, recombinantform, or chemically synthesized to include any type of nucleic acidanalog. The template nucleic acid can be linear, circular, or otherforms. The template nucleic acids can include an insert portion havingan insert sequence. The template nucleic acids can also include at leastone adaptor sequence. The insert portion can be isolated in any form,including chromosomal, genomic, organellar (e.g., mitochondrial,chloroplast or ribosomal), recombinant molecules, cloned, amplified,cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, wholegenomic DNA, obtained from fresh frozen paraffin embedded tissue, needlebiopsies, circulating tumor cells, cell free circulating DNA, or anytype of nucleic acid library. The insert portion can be isolated fromany source including from organisms such as prokaryotes, eukaryotes(e.g., humans, plants and animals), fungus, viruses cells, tissues,normal or diseased cells or tissues, body fluids including blood, urine,serum, lymph, tumor, saliva, anal and vaginal secretions, amnioticsamples, perspiration, semen, environmental samples, culture samples, orsynthesized nucleic acid molecules prepared using recombinant molecularbiology or chemical synthesis methods. The insert portion can beisolated from any organ, including head, neck, brain, breast, ovary,cervix, colon, rectum, endometrium, gallbladder, intestines, bladder,prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid,pituitary, thymus, skin, heart, larynx, or other organs. The templatenucleic acid can be subjected to nucleic acid analysis, includingsequencing and composition analysis.

The term “adaptor” and related terms refers to oligonucleotides that canbe operably linked to a target polynucleotide, where the adaptor confersa function to the co-joined adaptor-target molecule. Adaptors compriseDNA, RNA, chimeric DNA/RNA, or analogs thereof. Adaptors can include atleast one ribonucleoside residue. Adaptors can be single-stranded,double-stranded, or have single-stranded and/or double-strandedportions. Adaptors can be configured to be linear, stem-looped, hairpin,or Y-shaped forms. Adaptors can be any length, including 4-100nucleotides or longer. Adaptors can have blunt ends, overhang ends, or acombination of both. Overhang ends include 5′ overhang and 3′ overhangends. The 5′ end of a single-stranded adaptor, or one strand of adouble-stranded adaptor, can have a 5′ phosphate group or lack a 5′phosphate group. Adaptors can include a 5′ tail that does not hybridizeto a target polynucleotide (e.g., tailed adaptor), or adaptors can benon-tailed. An adaptor can include a sequence that is complementary toat least a portion of a primer, such as an amplification primer, asequencing primer, or a capture primer (e.g., soluble or immobilizedcapture primers). Adaptors can include a random sequence or degeneratesequence. Adaptors can include at least one inosine residue. Adaptorscan include at least one phosphorothioate, phosphorothiolate and/orphosphoramidate linkage. Adaptors can include a barcode sequence whichcan be used to distinguish polynucleotides (e.g., insert sequences) fromdifferent sample sources in a multiplex assay. Adaptors can include aunique identification sequence (e.g., unique molecular index, UMI; or aunique molecular tag) that can be used to uniquely identify a nucleicacid molecule to which the adaptor is appended. In some embodiments, aunique identification sequence can be used to increase error correctionand accuracy, reduce the rate of false-positive variant calls and/orincrease sensitivity of variant detection. Adaptors can include at leastone restriction enzyme recognition sequence, including any one or anycombination of two or more selected from a group consisting of type I,type II, type III, type IV, type Hs or type IIB.

In some embodiments, any of the amplification primer sequences,sequencing primer sequences, capture primer sequences, target capturesequences, circularization anchor sequences, sample barcode sequences,spatial barcode sequences, or anchor region sequences can be about 3-50nucleotides in length, or about 5-40 nucleotides in length, or about5-25 nucleotides in length.

The term “universal sequence” and related terms refers to a sequence ina nucleic acid molecule that is common among two or more polynucleotidemolecules. For example, an adaptor having a universal sequence can beoperably joined to a plurality of polynucleotides so that the populationof co-joined molecules carry the same universal adaptor sequence.Examples of universal adaptor sequences include an amplification primersequence, a sequencing primer sequence or a capture primer sequence(e.g., soluble or immobilized capture primers).

When used in reference to nucleic acid molecules, the terms “hybridize”or “hybridizing” or “hybridization” or other related terms refers tohydrogen bonding between two different nucleic acids to form a duplexnucleic acid. Hybridization also includes hydrogen bonding between twodifferent regions of a single nucleic acid molecule to form aself-hybridizing molecule having a duplex region. Hybridization cancomprise Watson-Crick or Hoogstein binding to form a duplexdouble-stranded nucleic acid, or a double-stranded region within anucleic acid molecule. The double-stranded nucleic acid, or the twodifferent regions of a single nucleic acid, may be wholly complementary,or partially complementary. Complementary nucleic acid strands need nothybridize with each other across their entire length. The complementarybase pairing can be the standard A-T or C-G base pairing, or can beother forms of base-pairing interactions. Duplex nucleic acids caninclude mismatched base-paired nucleotides.

When used in reference to nucleic acids, the terms “extend”,“extending”, “extension” and other variants, refers to incorporation ofone or more nucleotides into a nucleic acid molecule. Nucleotideincorporation comprises polymerization of one or more nucleotides intothe terminal 3′ OH end of a nucleic acid strand, resulting in extensionof the nucleic acid strand. Nucleotide incorporation can be conductedwith natural nucleotides and/or nucleotide analogs. Typically, but notnecessarily, nucleotide incorporation occurs in a template-dependentfashion. Any suitable method of extending a nucleic acid molecule may beused, including primer extension catalyzed by a DNA polymerase or RNApolymerase.

In some embodiments, any of the amplification primer sequences,sequencing primer sequences, capture primer sequences (captureoligonucleotides), target capture sequences, circularization anchorsequences, sample barcode sequences, spatial barcode sequences, oranchor region sequences can be about 3-50 nucleotides in length, orabout 5-40 nucleotides in length, or about 5-25 nucleotides in length.

The term “nucleotides” or “nucleic acid” and related terms refers to amolecule comprising an aromatic base, a five carbon sugar (e.g., riboseor deoxyribose), and at least one phosphate group. Canonical ornon-canonical nucleotides are consistent with use of the term. Thephosphate in some embodiments comprises a monophosphate, diphosphate, ortriphosphate, or corresponding phosphate analog. The term “nucleoside”refers to a molecule comprising an aromatic base and a sugar.Nucleotides and nucleosides can be non-labeled or labeled with adetectable reporter moiety. A “derivative” of a nucleic acid ornucleotide can be substantially similar nucleotide derived from thenucleotide, such as, for example, in an amplification reaction.

Nucleotides (and nucleosides) typically comprise a hetero cyclic baseincluding substituted or unsubstituted nitrogen-containing parentheteroaromatic ring which are commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants, oranalogs of the same. The base of a nucleotide (or nucleoside) is capableof forming Watson-Crick and/or Hoogstein hydrogen bonds with anappropriate complementary base. Exemplary bases include, but are notlimited to, purines and pyrimidines such as: 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-Δ²-isopentenyladenine(6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine,guanine (G), isoguanine, N²-dimethylguanine (dmG), 7-methylguanine(7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine andO⁶-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),5,6-dihydrothymine, O⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines;hydroxymethylcytosines; 5-methycytosines; base (Y); as well asmethylated, glycosylated, and acylated base moieties; and the like.Additional exemplary bases can be found in Fasman, 1989, in “PracticalHandbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press,Boca Raton, Fla.

Nucleotides (and nucleosides) typically comprise a sugar moiety, such ascarbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48),acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal ChemistryLetters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al.,1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No.5,558,991). The sugar moiety comprises: ribosyl; 2′-deoxyribosyl;3′-deoxyribosyl; 2′,3′-dideoxyribosyl; 2′,3′-didehydrodideoxyribosyl;2′-alkoxyribosyl; 2′-azidoribosyl; 2′-aminoribosyl; 2′-fluororibosyl;2′-mercaptoriboxyl; 2′-alkylthioribosyl; 3′-alkoxyribosyl;3′-azidoribosyl; 3′-aminoribosyl; 3′-fluororibosyl; 3′-mercaptoriboxyl;3′-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

In some embodiments, nucleotides comprise a chain of one, two or threephosphorus atoms where the chain is typically attached to the 5′ carbonof the sugar moiety via an ester or phosphoramide linkage. In someembodiments, the nucleotide is an analog having a phosphorus chain inwhich the phosphorus atoms are linked together with intervening O, S,NH, methylene or ethylene. In some embodiments, the phosphorus atoms inthe chain include substituted side groups including O, S or BH₃. In someembodiments, the chain includes phosphate groups substituted withanalogs including phosphoramidate, phosphorothioate, phosphordithioate,and O-methylphosphoroamidite groups.

The term “reporter moiety”, “reporter moieties” or related terms refersto a compound that generates, or causes to generate, a detectablesignal. A reporter moiety is sometimes called a “label”. Any suitablereporter moiety may be used, including luminescent, photoluminescent,electroluminescent, bioluminescent, chemiluminescent, fluorescent,phosphorescent, chromophore, radioisotope, electrochemical, massspectrometry, Raman, hapten, affinity tag, atom, or an enzyme. Areporter moiety generates a detectable signal resulting from a chemicalor physical change (e.g., heat, light, electrical, pH, saltconcentration, enzymatic activity, or proximity events). A proximityevent includes two reporter moieties approaching each other, orassociating with each other, or binding each other. It is well known toone skilled in the art to select reporter moieties so that each absorbsexcitation radiation and/or emits fluorescence at a wavelengthdistinguishable from the other reporter moieties to permit monitoringthe presence of different reporter moieties in the same reaction or indifferent reactions. Two or more different reporter moieties can beselected having spectrally distinct emission profiles, or having minimaloverlapping spectral emission profiles. Reporter moieties can be linked(e.g., operably linked) to nucleotides, nucleosides, nucleic acids,enzymes (e.g., polymerases or reverse transcriptases), or support (e.g.,surfaces).

A reporter moiety (or label) comprises a fluorescent label or afluorophore. Exemplary fluorescent moieties which may serve asfluorescent labels or fluorophores include, but are not limited tofluorescein and fluorescein derivatives such as carboxyfluorescein,tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein,fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein,fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide,carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine andrhodamine derivatives such as TRITC, TMR, lissamine rhodamine, TexasRed, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine,TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissaminerhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Redhydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS,AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivativessuch as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY530/550 C3 hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, CascadeBlue and derivatives such as Cascade Blue acetyl azide, Cascade Bluecadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide,Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide,Lucifer Yellow CH, cyanine and derivatives such as indolium basedcyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyaninedyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes,imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates andderivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates,Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Greendyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes,Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), orHermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof,or any combination thereof. Cyanine dyes may exist in either sulfonatedor non-sulfonated forms, and consist of two indolenin, benzo-indolium,pyridium, thiozolium, and/or quinolinium groups separated by apolymethine bridge between two nitrogen atoms. Commercially availablecyanine fluorophores include, for example, Cy3, (which may comprise1[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indoliumor1[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate),Cy5 (which may comprise1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-iumor1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate),and Cy7 (which may comprise1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indoliumor1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate),where “Cy” stands for ‘cyanine’, and the first digit identifies thenumber of carbon atoms between two indolenine groups. Cy2 which is anoxazole derivative rather than indolenin, and the benzo-derivatizedCy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the reporter moiety can be a FRET pair, such thatmultiple classifications can be performed under a single excitation andimaging step. As used herein, FRET may comprise excitation exchange(Forster) transfers, or electron-exchange (Dexter) transfers.

The terms “linked”, “joined”, “attached”, “appended” and variantsthereof comprise any type of fusion, bond, adherence or associationbetween any combination of compounds or molecules that is of sufficientstability to withstand use in the particular procedure. The procedurecan include but are not limited to: nucleotide binding; nucleotideincorporation; de-blocking (e.g., removal of chain-terminating moiety);washing; removing; flowing; detecting; imaging and/or identifying. Suchlinkage can comprise, for example, covalent, ionic, hydrogen,dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds orassociations involving van der Waals forces, mechanical bonding, and thelike. In some embodiments, such linkage occurs intramolecularly, forexample linking together the ends of a single-stranded ordouble-stranded linear nucleic acid molecule to form a circularmolecule. In some embodiments, such linkage can occur between acombination of different molecules, or between a molecule and anon-molecule, including but not limited to: linkage between a nucleicacid molecule and a solid surface; linkage between a protein and adetectable reporter moiety; linkage between a nucleotide and detectablereporter moiety; and the like. Some examples of linkages can be found,for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition(2008); Aslam, M., Dent, A., “Bioconjugation: Protein CouplingTechniques for the Biomedical Sciences”, London: Macmillan (1998);Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques forthe Biomedical Sciences”, London: Macmillan (1998).

When used in reference to nucleic acids, the terms “amplify”,“amplifying”, “amplification”, and other related terms include producingmultiple copies of an original polynucleotide template molecule, wherethe copies comprise a sequence that is complementary to the templatesequence, or the copies comprise a sequence that is the same as thetemplate sequence. In some embodiments, the copies comprise a sequencethat is substantially identical to a template sequence, or issubstantially identical to a sequence that is complementary to thetemplate sequence.

The term “support” as used herein refers to a substrate that is designedfor deposition of biological molecules or biological samples for assaysand/or analyses. Examples of biological molecules to be deposited onto asupport include nucleic acids (e.g., DNA, RNA), polypeptides,saccharides, lipids, a single cell or multiple cells. Examples ofbiological samples include but are not limited to saliva, phlegm, mucus,blood, plasma, serum, urine, stool, sweat, tears and fluids from tissuesor organs.

In some embodiments, the support is solid, semi-solid, or a combinationof both. In some embodiments, the support is porous, semi-porous,non-porous, or any combination of porosity. In some embodiments, thesupport can be substantially planar, concave, convex, or any combinationthereof. In some embodiments, the support can be cylindrical, forexample comprising a capillary or interior surface of a capillary.

In some embodiments, the surface of the support can be substantiallysmooth. In some embodiments, the support can be regularly or irregularlytextured, including bumps, etched, pores, three-dimensional scaffolds,or any combination thereof.

In some embodiments, the support comprises a bead having any shape,including spherical, hemi-spherical, cylindrical, barrel-shaped,toroidal, disc-shaped, rod-like, conical, triangular, cubical,polygonal, tubular or wire-like.

The support can be fabricated from any material, including but notlimited to glass, fused-silica, silicon, a polymer (e.g., polystyrene(PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),polycarbonate (PC), polypropylene (PP), polyethylene (PE), high densitypolyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefincopolymers (COC), polyethylene terephthalate (PET)), or any combinationthereof. Various compositions of both glass and plastic substrates arecontemplated.

The support can have a plurality (e.g., two or more) of nucleic acidtemplates immobilized thereon. The plurality of immobilized nucleic acidtemplates have the same sequence or have different sequences. In someembodiments, individual nucleic acid template molecules in the pluralityof nucleic acid templates are immobilized to a different site on thesupport. In some embodiments, two or more individual nucleic acidtemplate molecules in the plurality of nucleic acid templates areimmobilized to a site on the support.

When used in reference to support, the term “feature” refers to a regionon a support. In some embodiments, the feature is a region on a coatingwhich is layered on the support. In some embodiments, the feature is aregion on a low non-specific binding coating which is layered on asupport. A support or coating can have a plurality of regions (e.g.,features) located at different pre-determined locations on the supportor coating (FIG. 3, right). The different features on the support can beplaced at non-overlapping positions or at overlapping positions on thesupport. The features can be configured to have any shape, for examplecircular, ovular, square, rectangular, or polygonal. The features can bearranged in a grid pattern having rows and columns, or can be arrangedin a row or a column. In some embodiments, any given feature contains aplurality of capture oligonucleotides and/or a plurality ofcircularization oligonucleotides immobilized to the support or to thecoating. The plurality of features includes at least a first and secondfeature.

The term “array” refers to a support comprising a plurality of siteslocated at pre-determined locations on the support to form an array ofsites. The sites can be discrete and separated by interstitial regions.In some embodiments, the pre-determined sites on the support can bearranged in one dimension in a row or a column, or arranged in twodimensions in rows and columns. In some embodiments, the plurality ofpre-determined sites is arranged on the support in an organized fashion.In some embodiments, the plurality of pre-determined sites is arrangedin any organized pattern, including rectilinear, hexagonal patterns,grid patterns, patterns having reflective symmetry, patterns havingrotational symmetry, or the like. The pitch between different pairs ofsites can be that same or can vary. In some embodiments, the supportcomprises at least 10² sites, at least 10³ sites, at least 10⁴ sites, atleast 10⁵ sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸sites, at least 10⁹ sites, at least 10¹⁰ sites, at least 10¹¹ sites, atleast 10¹² sites, at least 10¹³ sites, at least 10¹⁴ sites, at least10¹⁵ sites, or more, where the sites are located at pre-determinedlocations on the support. In some embodiments, a plurality ofpre-determined sites on the support (e.g., 10²-10¹⁵ sites or more) areimmobilized with nucleic acid templates to form a nucleic acid templatearray. In some embodiments, the nucleic acid templates that areimmobilized at a plurality of pre-determined sites by hybridization toimmobilized surface capture primers, or the nucleic acid templates arecovalently attached to the surface capture primer. In some embodiments,the nucleic acid templates that are immobilized at a plurality ofpre-determined sites, for example immobilized at 10²-10¹⁵ sites or more.In some embodiments, the immobilized nucleic acid templates areclonally-amplified to generate immobilized nucleic acid clusters at theplurality of pre-determined sites. In some embodiments, individualimmobilized nucleic acid clusters comprise linear clusters, or comprisesingle-stranded or double-stranded concatemers.

In some embodiments, a support comprising a plurality of sites locatedat random locations on the support is referred to herein as a supporthaving randomly located sites thereon. The location of the randomlylocated sites on the support are not pre-determined. The plurality ofrandomly-located sites is arranged on the support in a disordered and/orunpredictable fashion. In some embodiments, the support comprises atleast 10² sites, at least 10³ sites, at least 10⁴ sites, at least 10⁵sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸ sites, atleast 10⁹ sites, at least 10¹⁰ sites, at least 10¹¹ sites, at least 10¹²sites, at least 10¹³ sites, at least 10¹⁴ sites, at least 10¹⁵ sites, ormore, where the sites are randomly located on the support. In someembodiments, a plurality of randomly located sites on the support (e.g.,10²-10¹⁵ sites or more) are immobilized with nucleic acid templates toform a support immobilized with nucleic acid templates. In someembodiments, the nucleic acid templates that are immobilized at aplurality of randomly located sites by hybridization to immobilizedsurface capture primers, or the nucleic acid templates are covalentlyattached to the surface capture primer. In some embodiments, the nucleicacid templates that are immobilized at a plurality of randomly locatedsites, for example immobilized at 10²-10¹⁵ sites or more. In someembodiments, the immobilized nucleic acid templates areclonally-amplified to generate immobilized nucleic acid clusters at theplurality of randomly located sites. In some embodiments, individualimmobilized nucleic acid clusters comprise linear clusters, or comprisesingle-stranded or double-stranded concatemers.

In some embodiment, the plurality of immobilized surface capture primerson the support are in fluid communication with each other to permitflowing a solution of reagents (e.g., nucleic acid template molecules,soluble primers, enzymes, nucleotides, divalent cations, buffers, andthe like) onto the support so that the plurality of immobilized surfacecapture primers on the support can be essentially simultaneously reactedwith the reagents in a massively parallel manner. In some embodiments,the fluid communication of the plurality of immobilized surface captureprimers can be used to conduct nucleic acid amplification reactions(e.g., RCA, MDA, PCR and bridge amplification) essentiallysimultaneously on the plurality of immobilized surface capture primers.

In some embodiment, the plurality of immobilized nucleic acid clusterson the support are in fluid communication with each other to permitflowing a solution of reagents (e.g., enzymes, nucleotides, divalentcations, and the like) onto the support so that the plurality ofimmobilized nucleic acid clusters on the support can be essentiallysimultaneously reacted with the reagents in a massively parallel manner.In some embodiments, the fluid communication of the plurality ofimmobilized nucleic acid clusters can be used to conduct nucleotidebinding assays and/or conduct nucleotide polymerization reactions (e.g.,primer extension or sequencing) essentially simultaneously on theplurality of immobilized nucleic acid clusters, and optionally toconduct detection and imaging for massively parallel sequencing.

When used in reference to immobilized enzymes, the term “immobilized”and related terms refer to enzymes (e.g., polymerases) that are attachedto a support through covalent bond or non-covalent interaction, orattached to a coating on the support, or buried within a matrix formedby a coating on the support.

When used in reference to immobilized nucleic acids, the term“immobilized” and related terms refer to nucleic acid molecules that areattached to a support through covalent bond or non-covalent interaction,or attached to a coating on the support, or buried within a matrixformed by a coating on the support, where the nucleic acid moleculesinclude surface capture primers, nucleic acid template molecules andextension products of capture primers. Extension products of captureprimers includes nucleic acid concatemers (e.g., nucleic acid clusters).

In some embodiments, one or more nucleic acid templates are immobilizedon the support, for example immobilized at the sites on the support. Insome embodiments, the one or more nucleic acid templates areclonally-amplified. In some embodiments, the one or more nucleic acidtemplates are clonally-amplified off the support (e.g., in-solution) andthen deposited onto the support and immobilized on the support. In someembodiments, the clonal amplification reaction of the one or morenucleic acid templates is conducted on the support resulting inimmobilization on the support. In some embodiments, the one or morenucleic acid templates are clonally-amplified (e.g., in solution or onthe support) using a nucleic acid amplification reaction, including anyone or any combination of: polymerase chain reaction (PCR), multipledisplacement amplification (MDA), transcription-mediated amplification(TMA), nucleic acid sequence-based amplification (NASBA), stranddisplacement amplification (SDA), real-time SDA, bridge amplification,isothermal bridge amplification, rolling circle amplification (RCA),circle-to-circle amplification, helicase-dependent amplification,recombinase-dependent amplification, and/or single-stranded binding(SSB) protein-dependent amplification.

The term “surface capture primer”, “capture oligonucleotide” and relatedterms refers to single-stranded oligonucleotides that are immobilized toa support and comprise a sequence that can hybridize to at least aportion of a nucleic acid template molecule. Surface capture primers canbe used to immobilize template molecules to a support via hybridization.Surface capture primers can be immobilized to a support in a manner thatresists primer removal during flowing, washing, aspirating, and changesin temperature, pH, salts, chemical and/or enzymatic conditions.Typically, but not necessarily, the 5′ end of a surface capture primercan be immobilized to a support. Alternatively, an interior portion orthe 3′ end of a surface capture primer can be immobilized to a support.

The sequence of surface capture primers can be wholly or partiallycomplementary along their length to at least a portion of the nucleicacid template molecule. A support can include a plurality of immobilizedsurface capture primers having the same sequence, or having two or moredifferent sequences. Surface capture primers can be any length, forexample 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides,or longer lengths.

A surface capture primer can have a terminal 3′ nucleotide having asugar 3′ OH moiety which is extendible for nucleotide polymerization(e.g., polymerase catalyzed polymerization). A surface capture primercan have a terminal 3′ nucleotide having the 3′ sugar position linked toa chain-terminating moiety that inhibits nucleotide polymerization. The3′ chain-terminating moiety can be removed (e.g., de-blocked) to convertthe 3′ end to an extendible 3′ OH end using a de-blocking agent.Examples of chain terminating moieties include alkyl group, alkenylgroup, alkynyl group, allyl group, aryl group, benzyl group, azidegroup, amine group, amide group, keto group, isocyanate group, phosphategroup, thio group, disulfide group, carbonate group, urea group, orsilyl group. Azide type chain terminating moieties including azide,azido and azidomethyl groups. Examples of de-blocking agents include aphosphine compound, such as Tris(2-carboxyethyl)phosphine (TCEP) andbis-sulfo triphenyl phosphine (BS-TPP), for chain-terminating groupsazide, azido and azidomethyl groups. Examples of de-blocking agentsinclude tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) withpiperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ),for chain-terminating groups alkyl, alkenyl, alkynyl and allyl. Examplesof a de-blocking agent includes Pd/C for chain-terminating groups aryland benzyl. Examples of de-blocking agents include phosphine,beta-mercaptoethanol or dithiothritol (DTT), for chain-terminatinggroups amine, amide, keto, isocyanate, phosphate, thio and disulfide.Examples of de-blocking agents include potassium carbonate (K₂CO₃) inMeOH, triethylamine in pyridine, and Zn in acetic acid (AcOH), forcarbonate chain-terminating groups. Examples of de-blocking agentsinclude tetrabutylammonium fluoride, pyridine-HF, with ammoniumfluoride, and triethylamine trihydrofluoride, for chain-terminatinggroups urea and silyl.

The term “branched polymer” and related terms refers to a polymer havinga plurality of functional groups that help conjugate a biologicallyactive molecule such as a nucleotide, and the functional group can beeither on the side chain of the polymer or directly attaches to acentral core or central backbone of the polymer. The branched polymercan have linear backbone with one or more functional groups coming offthe backbone for conjugation. The branched polymer can also be a polymerhaving one or more sidechains, wherein the side chain has a sitesuitable for conjugation. Examples of the functional group include butare limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde,aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activesulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, andtresylate.

When used in reference to a low binding surface coating, one or morelayers of a multi-layered surface coating may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(2-hydroxylethylmethacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol)methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid(branched PGA), branched poly-lysine, branched poly-glucoside, anddextran.

In some embodiments, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branched.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500, at least 1,000, at least 2,000, atleast 3,000, at least 4,000, at least 5,000, at least 10,000, at least15,000, at least 20,000, at least 25,000, at least 30,000, at least35,000, at least 40,000, at least 45,000, or at least 50,000 daltons.

In some embodiments, e.g., wherein at least one layer of a multi-layeredsurface comprises a branched polymer, the number of covalent bondsbetween a branched polymer molecule of the layer being deposited andmolecules of the previous layer may range from about one covalentlinkages per molecule and about 32 covalent linkages per molecule. Insome embodiments, the number of covalent bonds between a branchedpolymer molecule of the new layer and molecules of the previous layermay be at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 12,at least 14, at least 16, at least 18, at least 20, at least 22, atleast 24, at least 26, at least 28, at least 30, or at least 32 covalentlinkages per molecule.

Any reactive functional groups that remain following the coupling of amaterial layer to the surface may optionally be blocked by coupling asmall, inert molecule using a high yield coupling chemistry. Forexample, in the case that amine coupling chemistry is used to attach anew material layer to the previous one, any residual amine groups maysubsequently be acetylated or deactivated by coupling with a small aminoacid such as glycine.

The number of layers of low non-specific binding material, e.g., ahydrophilic polymer material, deposited on the surface, may range from 1to about 10. In some embodiments, the number of layers is at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10. In some embodiments, the number oflayers may be at most 10, at most 9, at most 8, at most 7, at most 6, atmost 5, at most 4, at most 3, at most 2, or at most 1. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someembodiments the number of layers may range from about 2 to about 4. Insome embodiments, all of the layers may comprise the same material. Insome embodiments, each layer may comprise a different material. In someembodiments, the plurality of layers may comprise a plurality ofmaterials. In some embodiments at least one layer may comprise abranched polymer. In some embodiment, all of the layers may comprise abranched polymer.

One or more layers of low non-specific binding material may in somecases be deposited on and/or conjugated to the substrate surface using apolar protic solvent, a polar or polar aprotic solvent, a nonpolarsolvent, or any combination thereof. In some embodiments the solventused for layer deposition and/or coupling may comprise an alcohol (e.g.,methanol, ethanol, propanol, etc.), another organic solvent (e.g.,acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),etc.), water, an aqueous buffer solution (e.g., phosphate buffer,phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS),etc.), or any combination thereof. In some embodiments, an organiccomponent of the solvent mixture used may comprise at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% of the total, with the balance made up of water oran aqueous buffer solution. In some embodiments, an aqueous component ofthe solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% of the total, with the balance made up of an organicsolvent. The pH of the solvent mixture used may be less than 6, about 6,6.5, 7, 7.5, 8, 8.5, 9, or greater than pH 9.

The term “persistence time” and related terms refers to the length oftime that a binding complex, which is formed between the target nucleicacid, a polymerase, a conjugated or unconjugated nucleotide, remainsstable without any binding component dissociates from the bindingcomplex. The persistence time is indicative of the stability of thebinding complex and strength of the binding interactions. Persistencetime can be measured by observing the onset and/or duration of a bindingcomplex, such as by observing a signal from a labeled component of thebinding complex. For example, a labeled nucleotide or a labeled reagentcomprising one or more nucleotides may be present in a binding complex,thus allowing the signal from the label to be detected during thepersistence time of the binding complex. One exemplary label is afluorescent label.

The hybridization buffers described herein comprise a first and secondpolar aprotic solvent, a pH buffer system and a crowding agent. Thepolar solvent as included in the hybridization composition describedherein is a solvent or solvent system comprising one or more moleculescharacterized by the presence of a permanent dipole moment, i.e., amolecule having a spatially unequal distribution of charge density. Apolar solvent may be characterized by a dielectric constant of 20, 25,30, 35, 40, 45, 50, 55, 60 or by a value or a range of values having anyof the aforementioned values. A polar solvent as described herein maycomprise a polar aprotic solvent. A polar aprotic solvent as describedherein may further contain no ionizable hydrogen in the molecule. Inaddition, polar solvents or polar aprotic solvents may be preferablysubstituted in the context of the presently disclosed compositions witha strong polarizing functional groups such as nitrile, carbonyl, thiol,lactone, sulfone, sulfite, and carbonate groups so that the underlyingsolvent molecules have a dipole moment. Polar solvents and polar aproticsolvents can be present in both aliphatic and aromatic or cyclic form.In some embodiments, the polar solvent is acetonitrile.

The polar or polar aprotic solvent described herein can have adielectric constant that is the same as or close to acetonitrile. Thedielectric constant of the polar or polar aprotic solvent can be in therange of about 20-60, about 25-55, about 25-50, about 25-45, about25-40, about 30-50, about 30-45, or about 30-40. The dielectric constantof the polar or polar aprotic solvent can be greater than 20, 25, 30,35, or 40. The dielectric constant of the polar or polar aprotic solventcan be lower than 30, 40, 45, 50, 55, or 60. The dielectric constant ofthe polar or polar aprotic solvent can be about 35, 36, 37, 38, or 39.

The polar or polar aprotic solvent described herein can have a polarityindex that is the same as or close to acetonitrile. The polarity indexof the polar or polar aprotic solvent can be in the range of about 2-9,2-8, 2-7, 2-6, 3-9, 3-8, 3-7, 3-6, 4-9, 4-8, 4-7, or 4-6. The polarityindex of the polar or polar aprotic solvent can be greater than about 2,3, 4, 4.5, 5, 5.5, or 6. The polarity index of the polar or polaraprotic solvent can be lower than about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, or 10. The polarity index of the polar or polar aprotic solventcan be about 5.5, 5.6, 5.7, or 5.8.

Some examples of the polar or polar aprotic solvent include but are notlimited to acetonitrile, dimethylformamide (DMF), dimethylsulfoxide(DMSO), acetanilide, N-acetyl pyrrolidone, 4-amino pyridine, benzamide,benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylenecarbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleicanhydride, 2-chlorocyclohexanone, chloroethylene carbonate,chloronitromethane, citraconic anhydride, crotonlactone,5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethylsulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, ethylene glycol sulfite, furfural,2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxybenzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate,1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.

The amount of the polar solvent or polar aprotic solvent is present inan amount effective to denature a double stranded nucleic acid. In someembodiments, the amount of the polar or polar aprotic solvent is greaterthan about 10% by volume based on the total volume of the formulation.The amount of the polar or polar aprotic solvent is about or more thanabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher, by volume based on the total volume of the formulation. Theamount of the polar or polar aprotic solvent is lower than about 15%,20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volumebased on the total volume of the formulation. In some embodiments, theamount of the polar or polar aprotic solvent is in the range of about10% to 90% by volume based on the total volume of the formulation. Insome embodiments, the amount of the polar or polar aprotic solvent is inthe range of about 25% to 75% by volume based on the total volume of theformulation. In some embodiments, the amount of the polar or polaraprotic solvent is in the range of about 10% to 95%, 10% to 85%, 20% to90%, 20% to 80%, 20% to 75%, or 30% to 60% by volume based on the totalvolume of the formulation.

In some embodiments, the disclosed hybridization buffer formulations mayinclude the addition of an organic solvent. Examples of suitablesolvents include, but are not limited to, acetonitrile, ethanol, DMF,and methanol, or any combination thereof at varying percentages(typically >5%). In some embodiments, the percentage of organic solvent(by volume) included in the hybridization buffer may range from about 1%to about 20%. In some embodiments, the percentage by volume of organicsolvent may be at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 15%, or at least 20%. In some embodiments, the percentageby volume of organic solvent may be at most 20%, at most 15%, at most10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most4%, at most 3%, at most 2%, or at most 1%. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of organic solvent may range from about 4% to about 15%. Those ofskill in the art will recognize that the percentage by volume of organicsolvent may have any value within this range, e.g., about 7.5%.

Improvements in hybridization rate: In some embodiments, the use ofoptimized buffer formulations disclosed herein (optionally, used incombination with low non-specific binding surface) yield relativehybridization rates that range from about 2× to about 20× faster thanthat for a conventional hybridization protocol. In some embodiments, therelative hybridization rate may be at least 2×, at least 3×, at least4×, at least 5×, at least 6×, at least 7×, at least 8×, at least 9×, atleast 10×, at least 12×, at least 14×, at least 16×, at least 18×, or atleast 20× that for a conventional hybridization protocol.

Improvements in hybridization efficiency (or yield) is a measure of thepercentage of total available tethered adapter sequences on a solidsurface, primer sequences, or oligonucleotide sequences in general thatare hybridized to complementary sequences. In some embodiments, the useof optimized buffer formulations disclosed herein (optionally, used incombination with low non-specific binding surface) yield improvedhybridization efficiency compared to that for a conventionalhybridization protocol. In some embodiments, the hybridizationefficiency that may be achieved is better than 80%, 85%, 90%, 95%, 98%,or 99% in any of the hybridization reaction times specified above.

Improvements in hybridization specificity is a measure of the ability oftethered adapter sequences, primer sequences, or oligonucleotidesequences in general to correctly hybridize only to completelycomplementary sequences. In some embodiments, the use of the optimizedbuffer formulations disclosed herein (optionally, used in combinationwith low non-specific binding surface) yield improved hybridizationspecificity compared to that for a conventional hybridization protocol.In some embodiments, the hybridization specificity that may be achievedis better than 1 base mismatch in 10 hybridization events, 1 basemismatch in 100 hybridization events, 1 base mismatch in 1,000hybridization events, or 1 base mismatch in 10,000 hybridization events.

The term “polymer-nucleotide conjugate,” or “multivalent molecule”, andrelated terms refers generally to a molecule comprising (a) a core, and(b) a plurality of nucleotide arms where each nucleotide arm comprises(i) a core attachment moiety, (ii) a spacer comprising a PEG moiety,(iii) a linker, and (iv) a nucleotide unit. In some embodiments, thepolymer-nucleotide conjugate comprises a core which is attached to theplurality of nucleotide arms. In some embodiments, the spacer isattached to the linker, wherein the linker is attached to the nucleotideunit. The multivalent comprise a polymer-nucleotide conjugate,comprising a plurality of copies of the same nucleotide attached to theparticle, wherein the plurality of nucleotides are each part of anucleotide arm. See for example FIGS. 5A-D and FIGS. 6A-C. When thenucleotide is complementary to the target nucleic acid, thepolymer-nucleotide conjugate forms a binding complex with the polymeraseand the target nucleic acid, and the binding complex exhibits increasedstability and longer persistence time than the binding complex formedusing a single unconjugated or untethered nucleotide. Compositions andmethods for preparing and using the multivalent molecules are describedin U.S. Ser. No. 16/579,794, filed on Sep. 23, 2019, the contents ofwhich is hereby expressly incorporated by reference in its entirety.

The term “multivalent binding complex” and related terms refersgenerally to a complex formed between a polymer-nucleotide conjugate andtwo or more nucleotides in two or more copies of a target nucleic acidsequence at substantially the same time, such as, for example, in asingle nucleotide binding reaction. The two or more copies of the targetnucleic acid sequence may be on the same target nucleic acid molecule(e.g., concatemer) or a different target nucleic acid molecule.

The term “crowding agent” and related terms refers to a compound thatalters the properties of other molecules in a solution. Crowding agentstypically have high molecular weight and/or bulky structures. Crowdingagents in solution can increase the concentration of other molecules inthe solution. Crowding agents can reduce the volume of solvent that isavailable for other molecules in the solution which can create amolecular crowding environment. Crowding agents in a solution cangenerate a crowded environment for molecules in the solution. Crowdingagents can alter the rates or equilibrium constants of a reaction.Examples of crowding agents include polyethylene glycol (e.g., PEG),ficoll, dextran, glycogen, polyvinyl alcohol, triblock polymers (e.g.,Pluronics), polystyrene, polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC),hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methycellulose,and hydroxyl methyl cellulose. In some embodiments, the crowding agentcomprises linear or branched PEG. In some embodiments, the crowdingagent comprise PEG 400, PEG 1500, PEG 2000, PEG 3400, PEG 3350, PEG4000, PEG 6000 or PEG 8000. In some embodiments, a solution can includeat least one crowding agent at about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 60%, or higher percent based on volume of thesolution. In some embodiments, the solution can be used for nucleic acidamplification including rolling circle amplification and/or multipledisplacement amplification reactions.

A suitable amount of a crowding agent in the composition allows for,enhances, or facilitates molecular crowding. The amount of the crowdingagent is about or more than about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 60%, or higher, by volume based on the total volumeof the formulation. In some cases, the amount of the molecular crowdingagent is greater than 5% by volume based on the total volume of theformulation. The amount of the crowding agent is lower than about 3%,5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%,or higher, by volume based on the total volume of the formulation. Insome cases, the amount of the molecular crowding agent can be less than30% by volume based on the total volume of the formulation. In someembodiments, the amount of the polar or polar aprotic solvent is in therange of about 25% to 75% by volume based on the total volume of theformulation. In some embodiments, the amount of the polar or polaraprotic solvent is in the range of about 1% to 40%, 1% to 35%, 2% to50%, 2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%,5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, byvolume based on the total volume of the formulation. In some cases, theamount of the molecular crowding agent can be in the range of about 5%to about 20% by volume based on the total volume of the formulation. Insome embodiments, the amount of the crowding agent is in the range ofabout 1% to 30% by volume based on the total volume of the formulation.

In some embodiments, the disclosed hybridization buffer formulations mayinclude the addition of a molecular crowding or volume exclusion agent.Molecular crowding or volume exclusion agents are typicallymacromolecules (e.g., proteins) which, when added to a solution in highconcentrations, may alter the properties of other molecules in solutionby reducing the volume of solvent available to the other molecules. Insome embodiments, the percentage by volume of molecular crowding orvolume exclusion agent included in the hybridization buffer formulationmay range from about 1% to about 50%. In some embodiments, thepercentage by volume of molecular crowding or volume exclusion agent maybe at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, orat least 50%. In some embodiments, the percentage by volume of molecularcrowding or volume exclusion agent may be at most 50%, at most 45%, atmost 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most15%, at most 10%, at most 5%, or at most 1%. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of molecular crowding or volume exclusion agent may range fromabout 5% to about 35%. Those of skill in the art will recognize that thepercentage by volume of molecular crowding or volume exclusion agent mayhave any value within this range, e.g., about 12.5%.

The hybridization buffer described herein includes a pH buffer systemthat maintains the pH of the compositions in a range suitable forhybridization process. The pH buffer system can include one or morebuffering agents selected from the group consisting of Tris, HEPES,TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, MES, and MOPS.The pH buffer system can further include a solvent. A preferred pHbuffer system includes MOPS, IVIES, TAPS, phosphate buffer combined withmethanol, acetonitrile, ethanol, isopropanol, butanol, t-butyl alcohol,DMF, DMSO, or any combination therein.

The hybridization buffer includes an amount of the pH buffer system thatis effective to maintain the pH of the formulation to be in a rangesuitable for the hybridization. In some embodiments, the pH may be atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10. In some embodiments, the pH may be at most 10,at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, or atmost 3. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, the pH of the hybridization buffer may range from about 4to about 8. Those of skill in the art will recognize that the pH of thehybridization buffer may have any value within this range, e.g., aboutpH 7.8. In some cases, the pH range is about 3 to about 10. In someembodiments, the disclosed hybridization buffer formulations may includeadjustment of pH over the range of about pH 3 to pH 10, with a preferredbuffer range of 5-9.

The hybridization buffer described herein includes an additive (e.g.,polar aprotic solvent) for controlling melting temperature of nucleicacid can vary depending on other agents used in the compositions. Theamount of the additive for controlling melting temperature of thenucleic acid is about or more than about 1%, 2%, 3%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 50%, 60%, or higher, by volume based on the totalvolume of the formulation. In some cases, the amount of the additive forcontrolling melting temperature of the nucleic acid is greater thanabout 2% by volume based on the total volume of the formulation. In somecases, the amount of the additive for controlling melting temperature ofthe nucleic acid is greater than 5% by volume based on the total volumeof the formulation. In some cases, the amount of the additive forcontrolling melting temperature of the nucleic acid is lower than about3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%,90%, or higher, by volume based on the total volume of the formulation.In some embodiments, the amount of the additive for controlling meltingtemperature of the nucleic acid is in the range of about 1% to 40%, 1%to 35%, 2% to 50%, 2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to20%, 2% to 10%, 5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%,5% to 20%, by volume based on the total volume of the formulation. Insome embodiments, the amount of the additive for controlling meltingtemperature of the nucleic acid is in the range of about 2% to 20% byvolume based on the total volume of the formulation. In some cases, theamount of the additive for controlling melting temperature of thenucleic acid is in the range of about 5% to 10% by volume based on thetotal volume of the formulation.

In some embodiments, the disclosed hybridization buffer formulations mayinclude the addition of an additive that alters nucleic acid duplexmelting temperature. Examples of suitable additives that may be used toalter nucleic acid melting temperature include, but are not limited to,Formamide. In some embodiments, the percentage by volume of a meltingtemperature additive included in the hybridization buffer formulationmay range from about 1% to about 50%. In some embodiments, thepercentage by volume of a melting temperature additive may be at least1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.In some embodiments, the percentage by volume of a melting temperatureadditive may be at most 50%, at most 45%, at most 40%, at most 35%, atmost 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most5%, or at most 1%. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, the percentage by volume of a meltingtemperature additive may range from about 10% to about 25%. Those ofskill in the art will recognize that the percentage by volume of amelting temperature additive may have any value within this range, e.g.,about 22.5%.

In some embodiments, the hybridization buffer described herein includesan additive that impacts DNA hydration: In some embodiments, thedisclosed hybridization buffer formulations may include the addition ofan additive that impacts nucleic acid hydration. Examples include, butare not limited to, betaine, urea, glycine betaine, or any combinationthereof. In some embodiments, the percentage by volume of a hydrationadditive included in the hybridization buffer formulation may range fromabout 1% to about 50%. In some embodiments, the percentage by volume ofa hydration additive may be at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50%. In some embodiments, thepercentage by volume of a hydration additive may be at most 50%, at most45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, atmost 15%, at most 10%, at most 5%, or at most 1%. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of a hydration additive may range from about 1% to about 30%.Those of skill in the art will recognize that the percentage by volumeof a melting temperature additive may have any value within this range,e.g., about 6.5%.

The term “sequencing” and related terms refers to a method for obtainingnucleotide sequence information from a nucleic acid molecule, typicallyby determining the identity of at least some nucleotides (includingtheir nucleobase components) within the nucleic acid molecule. In someembodiments, the sequence information of a given region of a nucleicacid molecule includes identifying each and every nucleotide within aregion that is sequenced. In some embodiments, sequencing informationdetermines only some of the nucleotides a region, while the identity ofsome nucleotides remains undetermined or incorrectly determined. Anysuitable method of sequencing may be used. In an exemplary embodiment,sequencing can include label-free or ion based sequencing methods. Insome embodiments, sequencing can include labeled or dye-containingnucleotide or fluorescent based nucleotide sequencing methods. In someembodiments, sequencing can include cluster-based sequencing or bridgesequencing methods.

In some embodiments, in any of the sequencing steps can be conductedusing a sequence-by-synthesis, sequence-by-hybridization orsequence-by-binding procedure. Examples of massively parallelsequence-by-synthesis procedures include polony sequencing,pyrosequencing (e.g., from 454 Life Sciences; U.S. Pat. Nos. 7,211,390,7,244,559 and 7,264,929), chain-terminator sequencing (e.g., fromIllumina; U.S. Pat. No. 7,566,537; Bentley 2006 Current Opinion Geneticsand Development 16:545-552; and Bentley, et al., 2008 Nature 456:53-59,ion-sensitive sequencing (e.g., from Ion Torrent), probe-anchor ligationsequencing (e.g., Complete Genomics), DNA nanoball sequencing, nanoporeDNA sequencing. Examples of single molecule sequencing include Heliscopesingle molecule sequencing, and single molecule real time (SMRT)sequencing. An example of sequence-by-hybridization includes SOLiDsequencing (e.g., from Life Technologies; WO 2006/084132). An example ofsequence-by-binding includes Omniome sequencing (e.g., U.S. Pat. No.10,246,744).

As used herein, “paired end” information refers to genetic sequenceinformation pertaining to both the forward and reverse strands of adouble stranded nucleic acid molecule or nucleic acid segment. Apaired-end read or paired-end sequencing thus refers to thedetermination of the sequence of both the forward and the reversestrand. This determination may be made directly and may in someembodiments be made without reference to the sequence of a knowncomplementary strand.

As used herein, the phrases “imaging module”, “imaging unit”, “imagingsystem”, “optical imaging module”, “optical imaging unit”, and “opticalimaging system” are used interchangeably, and may comprise components orsub-systems of a larger system that may also include, e.g., fluidicsmodules, temperature control modules, translation stages, robotic fluiddispensing and/or microplate handling, processor or computers,instrument control software, data analysis and display software, etc.

As used herein, the term “detection channel” refers to an optical path(and/or the optical components therein) within an optical system that isconfigured to deliver an optical signal arising from a sample to adetector. In some instances, a detection channel may be configured forperforming spectroscopic measurements, e.g., monitoring a fluorescencesignal or other optical signal using a detector such as aphotomultiplier. In some instances, a “detection channel” may be an“imaging channel”, i.e., an optical path (and/or the optical componentstherein) within an optical system that is configured to capture anddeliver an image to an image sensor.

As used herein, a “detectable label” may refer to any of a variety ofdetectable labels or tags known to those of skill in the art. Examplesinclude, but are not limited to, chromophores, fluorophores, quantumdots, upconverting phosphors, luminescent or chemiluminescent molecules,radioisotopes, magnetic nanoparticles, mass tags, and the like. In someinstances, a preferred label may comprise a fluorophore.

As used herein, the term “excitation wavelength” refers to thewavelength of light used to excite a fluorescent indicator (e.g., afluorophore or dye molecule) and generate fluorescence. Although theexcitation wavelength is typically specified as a single wavelength,e.g., 620 nm, it will be understood by those of skill in the art thatthis specification refers to a wavelength range or excitation filterbandpass that is centered on the specified wavelength. For example, insome instances, light of the specified excitation wavelength compriseslight of the specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm,±80 nm, or more. In some instances, the excitation wavelength used mayor may not coincide with the absorption peak maximum of the fluorescentindicator.

As used herein, the term “emission wavelength” refers to the wavelengthof light emitted by a fluorescent indicator (e.g., a fluorophore or dyemolecule) upon excitation by light of an appropriate wavelength.Although the emission wavelength is typically specified as a singlewavelength, e.g., 670 nm, it will be understood by those of skill in theart that this specification refers to a wavelength range or emissionfilter bandpass that is centered on the specified wavelength. In someinstances, light of the specified emission wavelength comprises light ofthe specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm,or more. In some instances, the emission wavelength used may or may notcoincide with the emission peak maximum of the fluorescent indicator.

As used herein, fluorescence is ‘specific’ if it arises fromfluorophores that are annealed or otherwise tethered to the surface,such as fluorescently labeled nucleic acid sequences having a region ofreverse complementarity to a corresponding segment of an oligonucleotideadapter on the surface and annealed to said corresponding segment. Thisfluorescence is contrasted with fluorescence arising from fluorophoresnot tethered to the surface through such an annealing process, or insome cases to background florescence of the surface.

The term “simple cell media” or related terms refers to a cell mediathat typically lacks ingredients to support cell growth and/orproliferation in culture. Simple cell media can be used for example towash, suspend, or dilute the cellular biological sample. Simple cellmedia can be mixed with certain ingredients to prepare a cell media thatcan support cell growth and/or proliferation in culture. A simple cellmedia comprises any one or any combination of two or more of a buffer, aphosphate compound, a sodium compound, a potassium compound, a calciumcompound, a magnesium compound and/or glucose. In some embodiments, thesimple cell media comprises PBS (phosphate buffered saline), DPBS(Dulbecco's phosphate-buffered saline), HBSS (Hank's balanced saltsolution), DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle'sMinimum Essential Medium), and/or EBSS. In some embodiments, thecellular biological sample or single cell can be placed in a simple cellmedia prior to or during the step of conducting any of the nucleic acidmethods described herein.

The term “complex cell media” or related terms refers to a cell mediathat can be used to support cell growth and/or proliferation in culturewithout supplementation or additives. Complex cell media can include anycombination of two or more of a buffering system (e.g., HEPES),inorganic salt(s), amino acid(s), protein(s), polypeptide(s),carbohydrate(s), fatty acid(s), lipid(s), purine(s) and theirderivatives (e.g., hypoxanthine), pyrimidine(s) and their derivatives,and/or trace element(s). Complex cell media includes fluids obtainedfrom a biological fluid or tissue extract. Complex cell media includesartificial cell media. In some embodiments, complex cell media can be aserum-containing media, for example complex cell media includesbiological fluids such as fetal bovine serum, blood plasma, blood serum,lymph fluid, human placental cord serum and amniotic fluid. In someembodiments, complex cell media can be a serum-free media, which aretypically (but not necessarily) defined cell culture media. In someembodiments, complex cell media can be a chemically-defined media whichtypically (but not necessarily) include recombinant polypeptides, andultra-pure inorganic and/or organic compounds. In some embodiments,complex cell media can be a protein-free media which include for exampleMEM (minimal essential media) and RPMI-1640 (Roswell Park MemorialInstitute). In some embodiments, the complex cell media comprises IMDM(Iscove's Modified Dulbecco's Medium. In some embodiments, the complexcell media comprises DMEM (Dulbecco's Modified Eagle's Medium). In someembodiments, the cellular biological sample or single cell can be placedin a complex cell media prior to or during the step of conducting any ofthe nucleic acid methods described herein.

The term “padlock probe” refers to a nucleic acid probe that typicallycomprises a linear single oligonucleotide strand that is designed tocapture target nucleic acid molecules by hybridization. Thehybridization complex can be circularized, and the circular molecule canbe subjected to a rolling circle amplification reaction for single-plexor multi-plex molecular detection methods. The padlock probe includestarget capture sequences at its 5′ terminal-end and 3′ terminal-end thatare complementary to contiguous regions of the target nucleic acidmolecule. The padlock probe can also include any one or any combinationof two or more adaptor sequences including an amplification primerbinding sequence, a sequencing primer binding sequence, animmobilization sequence and/or a sample index sequence. The variousadaptor sequences can be located in any region, for example the internalportion of the padlock probe. The 5′ and 3′ ends of the padlock probecan hybridize to adjacent positions on the target nucleic acid moleculeto form an open circularized molecule with a nick between the hybridized5′ and 3′ ends. The nick can be ligated to generate a covalently closecircular molecule. Alternatively, the 5′ and 3′ ends of the padlockprobe can hybridize to adjacent positions on the target nucleic acidmolecule to form an open circularized molecule with a gap between thehybridized 5′ and 3′ ends. The gap can be subject to apolymerase-mediated filled-in reaction to form a nick, and the nick canbe ligated to generate a covalently close circular molecule. Thecovalently closed circular molecule can be subjected to a rolling circleamplification reaction to generate a concatemer having tandem repeatregions containing the target sequence. The specificity for capturingthe target molecule in a mixture of target and non-target molecules isafforded by the requirement for specific hybridization of the 5′ and 3′ends to adjacent positions of the target molecule of interest to form anick, and enzymatically closing the nick which is only possible when the5′ and 3′ ends of the padlock probe have the correct basecomplementarity with the target molecule. A ligase enzyme thatdiscriminates between matched and mis-matched ends can be used to ensuresequence-specific hybridization. Thus, the covalently closed circularmolecule is formed if the target nucleic acid is present in the samplebeing tested.

Compositions and methods using the compositions for padlock-probe basedrolling circle amplification reactions are described in U.S. 63/059,723,filed on Jul. 31, 2020, the contents of which is hereby expresslyincorporated by reference in its entirety.

The term rolling circle amplification generally refers to anamplification method that employs a circularized nucleic acid templatemolecule containing a target sequence of interest, an amplificationprimer binding sequence, and optionally one or more adaptor sequencessuch as a sequencing primer binding sequence and/or a barcode. Therolling circle amplification reaction can be conducted under isothermalamplification conditions, and includes the circularized nucleic acidtemplate molecule, an amplification primer, a strand-displacingpolymerase and a plurality of nucleotides, to generate a concatemercontaining tandem repeat sequences of the circular template molecule andany adaptor sequences present in the original circularized nucleic acidtemplate molecule. The concatemer can self-collapse to form a nucleicacid nanoball. The shape and size of the nanoball can be furthercompacted by including a pair of inverted repeat sequences in thecircular template molecule, or by conducting the rolling circleamplification reaction with one or more compaction oligonucleotides. Oneof the advantages of using rolling circle amplification to generateclonal amplicons for a sequencing workflow, is that the repeat copies ofthe target sequence in the nanoball can be simultaneously sequenced toincrease signal intensity.

Kits. Provided here are kits useful for carrying out the methodsdisclosed herein using the systems and compositions disclosed herein. Akit may comprise a detectable polymer-nucleotide conjugate comprising:(i) a polymer core; and (ii) two or more nucleotide moieties attached tosaid polymer core. The kits described herein may have at least one, two,three, or four different types of detectable polymer-nucleotideconjugate, for example, in which each type of detectablepolymer-nucleotide conjugate has a different nucleotide moiety. The kitmay have a substrate comprising a surface having coupled thereto apolymer layer suitable to immobilize a biological sample or derivativethereof to said surface. In some kits, the biological sample (e.g., cellor tissue) is included in the kit. In some kits, the biological sampleis not included in the kit. The kit may comprise a hybridization bufferdisclosed herein, for example, comprising (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; and/or (ii) a second polar aproticsolvent having a dielectric constant that is less than or equal to 115.Optionally, capture oligonucleotides or components thereof, in situamplification reagents (e.g., buffers, primers, detectable labels), orcombinations thereof are included in the kit.

Instructions may be provided in the kits described herein, includinginstructions for hybridizing at least a portion of said target nucleicacid sequence to at least a portion of a capture oligonucleotide coupledto said surface. The kit may also comprise instructions for identifyingat least a portion of the target nucleic acid sequence within thebiological sample or derivative thereof by contacting said detectablepolymer-nucleotide conjugate with said biological sample or derivativethereof (e.g., containing the target nucleic acid molecule) underconditions sufficient to form a multivalent binding complex between saidtwo or more nucleotide moieties and said target nucleic acid sequence.

The kit may also comprise instructions for identifying at least aportion of a sub-cellular component within a cell or tissue in situ bycontacting said detectable polymer-nucleotide conjugate with saidsub-cellular component under conditions sufficient to form a multivalentbinding complex between said two or more nucleotide moieties and saidsub-cellular component.

Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia. The materials or componentsassembled in the kit can be provided to the practitioner stored in anyconvenient and suitable ways that preserve their operability andutility. For example the components can be in dissolved, dehydrated, orlyophilized form; they can be provided at room, refrigerated or frozentemperatures. The components are typically contained in suitablepackaging material(s). As employed herein, the phrase “packagingmaterial” refers to one or more physical structures used to house thecontents of the kit, such as compositions and the like. The packagingmaterial is constructed by well-known methods, preferably to provide asterile, contaminant-free environment. The packaging materials employedin the kit are those customarily utilized in gene expression assays andin the administration of treatments. As used herein, the term “package”refers to a suitable solid matrix or material such as glass, plastic,paper, foil, and the like, capable of holding the individual kitcomponents. Thus, for example, a package can be a glass vial orprefilled syringes used to contain suitable quantities of thepharmaceutical composition. The packaging material has an external labelwhich indicates the contents and/or purpose of the kit and itscomponents.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

II. EXEMPLARY EMBODIMENTS

Among the exemplary embodiments are:

-   -   1. A support comprising:    -   (a) a substrate coated with at least one hydrophilic polymer        coating having a water contact angle of no more than 45 degrees;    -   (b) a first feature comprising a first region of the hydrophilic        coating having immobilized thereon (1) a first plurality of        capture oligonucleotides which can hybridize to a plurality of a        first target nucleic acid molecules, and optionally, (2) a first        plurality of circularization oligonucleotides which can        circularize a captured first target nucleic acid molecule; and        optionally, (c) a second feature comprising a second region of        the hydrophilic coating having immobilized thereon (1) a second        plurality of capture oligonucleotides which can hybridize to a        plurality of a second target nucleic acid molecules, and (2) a        second plurality of circularization oligonucleotides which can        circularize a captured second target nucleic acid molecule.    -   2. The support of embodiment 1, wherein the support further        comprises a biological sample placed in contact with the first        and second features, wherein the biological sample comprises a        tissue, a plurality of cells or a single cell.    -   3. The support of embodiment 2, wherein the single cell, cells        in the tissue or the plurality of cells can be intact or        permeabilized or lysed.    -   4. The support of embodiments 1-2, wherein the support further        comprises a first target nucleic acid molecule hybridized to the        first target capture region in the first feature, and a second        target nucleic acid molecule hybridized to the second target        capture region in the second feature.    -   5. The support of embodiment 4, wherein the first and second        target nucleic acid molecules comprise DNA or RNA.    -   6. The support of embodiments 1-5, wherein a fluorescent image        of the support exhibits a contrast to noise ratio (CNR) of at        least 20.    -   7. The support of embodiments 1-6, wherein the hydrophilic        polymer coating can comprise at least one hydrophilic polymer        coating comprising a molecule selected from the group consisting        of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA),        poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP),        poly(acrylic acid) (PAA), polyacrylamide,        poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)        (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),        poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),        polyglutamic acid (PGA), poly-lysine, poly-glucoside,        streptavidin, and dextran.    -   8. The support of embodiment 7, wherein at least one layer of        the hydrophilic polymer coating comprises polyethylene glycol        (PEG).    -   9. The support of embodiments 1-8, wherein the substrate is        coated with a second hydrophilic polymer coating.    -   10. The support of embodiments 1-9, wherein the at least one        hydrophilic polymer coating comprises a polymer having a        molecular weight of at least 1,000 Daltons.    -   11. The support of embodiments 1-10, wherein the at least one        hydrophilic polymer coating comprises a branched hydrophilic        polymer having at least 4 branches.    -   12. The support of embodiments 1-11, wherein the at least one        hydrophilic polymer coating comprises: (a) a first layer        comprising a first monolayer of polymer molecules tethered to        the surface; (b) a second layer comprising a second monolayer of        polymer molecules tethered to the first monolayer of polymer        molecules; and (c) a third layer comprising a third monolayer of        polymer molecules tethered to the second monolayer of polymer        molecules, wherein the polymer molecules of the first layer, the        second layer or the third layer comprises branched polymer        molecules.    -   13. The support of embodiments 1-12, wherein the support can be        glass or plastic.    -   14. The support of embodiments 1-13, wherein the support can be        a planar support or a bead.    -   15. The support of embodiments 1-14, wherein the first plurality        of capture oligonucleotides and the first plurality of        circularization oligonucleotides in the first feature, and the        second plurality of capture oligonucleotides and the second        plurality of circularization oligonucleotides in the second        feature, are in fluid communication with each other so that the        first and second capture oligonucleotides and the first and        second circularization oligonucleotide react with reagents        (e.g., enzymes including polymerases, polymer-nucleotide        conjugates, nucleotides and/or divalent cations) in a massively        parallel manner.    -   16. The support of embodiments 1-15, wherein the support further        comprises a hybridization buffer comprising:        -   (i) a first polar aprotic solvent having a dielectric            constant that is no greater than 40 and having a polarity            index of 4-9;        -   (ii) a second polar aprotic solvent having a dielectric            constant that is no greater than 115 and is present in the            hybridization buffer formulation in an amount effective to            denature double-stranded nucleic acids;        -   (iii) a pH buffer system that maintains the pH of the            hybridization buffer formulation in a range of about 4-8;            and        -   (iv) a crowding agent in an amount sufficient to enhance or            facilitate molecular crowding.    -   17. The support of embodiment 16, wherein the first polar        aprotic solvent comprises acetonitrile at 25-50% by volume of        the hybridization buffer.    -   18. The support of embodiments 16-17, wherein the second polar        aprotic solvent comprises formamide at 5-10% by volume of the        hybridization buffer.    -   19. The support of embodiments 16-18, wherein the pH buffer        system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a        pH of 5-6.5.    -   20. The support of embodiments 16-19, wherein the crowding agent        comprises polyethylene glycol (PEG) at 5-35% by volume of the        hybridization buffer.    -   21. The support of embodiments 16-20, wherein the hybridization        buffer further comprises betaine.    -   22. The support of embodiments 1-21, wherein the support further        comprises at least one polymer-nucleotide conjugate comprising        two or more duplicates of a nucleotide moiety that are connected        to a core via a linker.    -   23. The support of embodiment 22, wherein the polymer-nucleotide        conjugate comprises:        -   (a) a core; and        -   (b) a plurality of nucleotide arms which comprise:            -   (i) a core attachment moiety;            -   (ii) a spacer comprising a PEG moiety;            -   (iii) a linker; and            -   (iv) a nucleotide unit.    -   24. The support of embodiments 1-23, wherein the first plurality        of capture oligonucleotides comprises:        -   (a) a first target capture region that hybridizes to at            least a portion of a first target nucleic acid molecule;        -   (b) a first universal sequence region comprising a first            spatial barcode sequence and, optionally, a first sample            barcode sequence;        -   (c) a first circularization anchor sequence; or        -   (d) a first cleavable region, and the first feature having            immobilized thereon, or any combination thereof    -   25. The support of embodiments 1-24, wherein the second        plurality of capture oligonucleotides comprises:        -   (a) a second target capture region that hybridizes to at            least a portion of a second target nucleic acid molecule;        -   (b) a second universal sequence region comprising a second            spatial barcode sequence, and optionally, a second sample            barcode sequence, and a second circularization anchor            sequence; and        -   (c) a second cleavable region, and the second feature having            immobilized thereon.    -   26. The support of embodiments 1-25, wherein the second        plurality of circularization oligonucleotides comprise:    -   (a) a second homopolymer region, and    -   (b) a second universal sequence region comprising a second        sequencing primer binding sequence and a second circularization        anchor binding sequence.    -   27. The support of embodiments 1-26, wherein the first plurality        of circularization oligonucleotides comprise:    -   (a) a first homopolymer region, and    -   (b) a first universal sequence region comprising a first        sequencing primer binding sequence and a first circularization        anchor binding sequence.    -   28. The support of embodiments 24-27, wherein the first and        second target capture regions of the first and second capture        oligonucleotides each comprise a random nucleotide sequence or a        target-specific nucleotide sequence.    -   29. The support of embodiments 24-28, wherein the first target        capture region in the first feature has the same sequence as the        second target capture region in the second feature.    -   30. The support of embodiments 24-29, wherein the first spatial        barcode sequence in the first feature has a different sequence        compared to the second spatial barcode sequence in the second        feature.    -   31. The support of embodiments 24-30, wherein the first sample        barcode sequence in the first feature has the same sequence as        the second sample barcode sequence in the second feature.    -   32. The support of embodiments 24-30, wherein the first        circularization anchor sequence in the first feature has the        same nucleotide sequence as the second circularization anchor        sequence in the second feature.    -   33. The support of embodiments 24-32, wherein the first        cleavable region in the first feature is cleavable with an        enzyme, a chemical compound, light or heat.    -   34. The support of embodiments 24-33, wherein the first        cleavable region is cleavable with the same condition as the        second cleavable region in the second feature.    -   35. The support of embodiments 24-34, wherein the first        homopolymer region of the first circularization oligonucleotide        in the first feature has the same sequence as the second        homopolymer region of the second circularization oligonucleotide        in the second feature.    -   36. The support of embodiments 24-35, wherein the first        sequencing primer binding sequence in the first feature has the        same sequence as the second sequencing primer binding sequence        in the second feature.    -   37. The support of embodiments 24-36, the first circularization        anchor binding sequence in the first feature has the same        sequence as the second circularization anchor binding sequence        in the second feature.    -   38. The support of embodiments 24-37, further comprising a first        primer extension product extended from the first target capture        region, wherein the first extension product comprises a        complementary sequence of at least a portion of the first target        nucleic acid molecule.    -   39. The support of embodiments 24-37, further comprising a        second primer extension product extended from the second target        capture region where the second extension product comprises a        complementary sequence of at least a portion of the second        target nucleic acid molecule.    -   40. The support of embodiments 23-39, wherein the core is        attached to the plurality of nucleotide arms.    -   41. The support of embodiments 23-40, wherein the spacer is        attached to the linker.    -   42. The support of embodiments 23-41, wherein the linker is        attached to the nucleotide unit.    -   43. The support of embodiments 23-42, wherein the nucleotide        unit comprises a base, sugar and at least one phosphate group.    -   44. The support of embodiments 23-43, wherein the linker is        attached to the nucleotide unit through the base.    -   45. The support of embodiments 23-44, wherein the linker        comprises an aliphatic chain or an oligo ethylene glycol chain        where both linker chains having 2-6 subunits and, optionally,        the linker includes an aromatic moiety.    -   46. The support of embodiments 23-45, wherein the plurality of        nucleotide arms have the same type of nucleotide unit which is        selected from a group consisting of dATP, dGTP, dCTP, dTTP and        dUTP.    -   47. The support of embodiments 23-45, wherein the plurality of        nucleotide arms have two or more different types of nucleotides        selected from a group consisting of dATP, dGTP, dCTP, dTTP and        dUTP.    -   48. The support of embodiments 23-47, wherein the nucleotide        unit has a chain terminating moiety (e.g., blocking moiety) at        the sugar 2′ position, at the sugar 3′ position, or at the sugar        2′ and 3′ position.    -   49. The support of embodiment 48, wherein the chain terminating        moiety is selected from a group consisting of 3′-deoxy        nucleotides, 2′,3′-di deoxynucleotides, 3′-methyl, 3′-azido,        3′-azidomethyl, 3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl,        3′-O-fluoroalkyl, 3′-fluoromethyl, 3′-difluoromethyl,        3′-trifluoromethyl, 3′-sulfonyl, 3′-malonyl, 3′-amino,        3′-O-amino, 3′-sulfhydral, 3′-aminomethyl, 3′-ethyl, 3′butyl,        3′-tert butyl, 3′-Fluorenylmethyloxycarbonyl, 3′        tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,        3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof    -   50. The support of embodiment 49, wherein the chain terminating        moiety is cleavable/removable from the nucleotide arm.    -   51. The support of embodiments 23-50, wherein the core is        labeled with detectable reporter moiety.    -   52. The support of embodiment 51, wherein the detectable        reporter moiety comprises a fluorophore.    -   53. The support of embodiments 23-52, wherein the core comprises        an avidin-like moiety and the core attachment moiety comprises        biotin.    -   54. A method for conducting cellularly addressable sequencing        and for analyzing nucleic acids from a biological sample,        comprising:        -   (a) providing a support comprising a low non-specific            binding coating to which an oligonucleotide suitable for            capturing/hybridizing a target nucleic acid molecule is            attached;        -   (b) contacting a target nucleic acid molecule with the            oligonucleotide under a buffer condition suitable to allow            the oligonucleotide to capture the target nucleic acid            molecule;        -   (c) optionally, amplifying the target nucleic acid molecule            to form an amplified nucleic acid product comprising a            linear single-stranded nucleic acid molecule comprising two            or more copies of the target nucleic acid sequence;        -   (d) contacting the amplified nucleic acid product with two            or more polymerases, and two or more sequencing primers that            hybridize to one or more regions of the amplified nucleic            acid product;        -   (e) contacting the amplified nucleic acid product with a            polymer-nucleotide conjugate comprising a polymer-nucleotide            conjugate under a condition suitable for forming a binding            complex between the amplified nucleic acid product and the            polymer-nucleotide conjugate, wherein the polymer-nucleotide            conjugate comprises two or more copies of a nucleotide and            (optionally) one or more detectable reporter moieties; and        -   (f) detecting the binding complex thereby identifying the            nucleotide base in the target nucleic acid molecule, wherein            the target nucleic acid molecule originates from a            biological tissue, and wherein the target nucleic acid            molecule is captured on the support in a manner so as to            preserve information related to the location of the cellular            origin of the target nucleic acid molecule.    -   55. The method of embodiment 54, wherein the oligonucleotide        comprises a capture oligonucleotide comprising:        -   (a) a target capture region that hybridizes to at least a            portion of a target nucleic acid molecule;        -   (b) a universal sequence region comprising a spatial barcode            sequence;        -   (c) a circularization anchor sequence configured to bind to            a circularization oligonucleotide; and        -   (d) a cleavable region.    -   56. The method of embodiment 55, wherein the circularization        oligonucleotide comprises:        -   (a) a homopolymer region;        -   (b) a universal sequence region comprising a sequencing            primer binding sequence; and        -   (c) a circularization anchor binding sequence; and    -   57. The method of embodiments 54-56, wherein the low        non-specific binding coating comprises at least one hydrophilic        polymer coating having a water contact angle of no more than 45        degrees.    -   58. The method of embodiments 54-57, wherein contacting in (b)        comprises hybridizing at least a portion of the target nucleic        acid molecule to the target capture region of the immobilized        capture oligonucleotide thereby forming an immobilized nucleic        acid duplex.    -   59. The method of embodiments 54-58, wherein optionally        amplifying in (c) comprises:        -   (a) conducting a primer extension reaction on the            immobilized nucleic acid duplex using the hybridized target            nucleic acid molecule as a template thereby forming an            immobilized target extension product;        -   (b) conducting a non-template tailing reaction on the            immobilized target extension product under conditions            suitable for appending a homopolymer tail to the target            extension product thereby forming an immobilized tailed            target extension product;        -   (c) cleaving the immobilized tailed target extension product            to release the immobilized tailed target extension product            from the low binding coating thereby forming a soluble            tailed target extension product;        -   (d) binding the soluble tailed target extension product to            one of a circularization oligonucleotide immobilized to the            low binding coating under a condition suitable to hybridize            the appended homopolymer tail of the soluble tailed target            extension product to the homopolymer region of the            immobilized circularization oligonucleotide, and suitable to            hybridize the circularization anchor sequence of the soluble            tailed target extension product to the circularization            anchor binding sequence of the immobilized circularization            oligonucleotide thereby forming an open circularized target            extension product with a gap;        -   (e) conducting a gap-filling primer extension reaction and a            ligation reaction on the open circularized target extension            product thereby forming a closed circular target extension            product which is hybridized to the immobilized            circularization oligonucleotide having a homopolymer region            with a 3′ extendible end; and        -   (f) conducting a rolling circle amplification reaction using            the 3′ extendible end of the homopolymer region under a            condition suitable to form an immobilized concatemer            molecule having tandem repeat regions comprising the            sequencing primer binding sequence, the target sequence, and            the spatial barcode sequence.    -   60. The method of embodiment 62, wherein determining the        sequence of the immobilized concatemer molecule comprises        sequencing the target sequence and the spatial barcode sequence.    -   61. The method of embodiments 54-60, wherein the target nucleic        acid molecule is RNA.    -   62. The method of embodiments 54-60, wherein the target nucleic        acid molecule is DNA.    -   63. A method for analyzing nucleic acids from a biological        sample, comprising:        -   (a) providing a support comprising a low non-specific            binding coating to which a plurality of capture            oligonucleotides are immobilized, wherein the plurality of            capture oligonucleotides comprise (i) a target capture            region (e.g., having a homopolymer sequence e.g., poly-T)            that hybridizes to at least a portion of a target RNA            molecule, (ii) a universal sequence region comprising a            spatial barcode sequence, and (iii) a cleavable region, and            wherein the low non-specific binding coating comprises at            least one hydrophilic polymer coating having a water contact            angle of no more than 45 degrees;        -   (b) contacting the low non-specific binding coating with a            target nucleic acid molecule under a condition (e.g., a            hybridization buffer) suitable for hybridizing at least a            portion of the target nucleic acid molecule to the target            capture region of one of the immobilized capture            oligonucleotides thereby forming an immobilized nucleic acid            duplex;        -   (c) conducting a primer extension reaction (e.g., reverse            transcription) on the immobilized nucleic acid duplex using            the hybridized target nucleic acid molecule as a template            thereby forming an immobilized target extension product;        -   (d) appending a nucleic acid adaptor to the immobilized            target extension product thereby forming an immobilized            adaptor-target extension product, wherein the nucleic acid            adaptor comprises a sequencing primer binding sequence;        -   (e) contacting the low non-specific binding coating with a            plurality of soluble circularization oligonucleotides under            a condition suitable for immobilizing at least one of            soluble circularization oligonucleotides to the low            non-specific binding coating in proximity to the immobilized            adaptor-target extension product, wherein the each of the            soluble circularization oligonucleotides in the plurality            comprises (i) an adaptor binding region (e.g., having a            sequencing primer binding sequence and optionally an            amplification primer binding sequence), (ii) a homopolymer            region, (iii) an anchor region, and (iv) an anchor moiety;        -   (f) hybridizing the target capture region (e.g., homopolymer            poly-T) of the immobilized adaptor-target extension product            to the homopolymer region of the immobilized circularization            oligonucleotide thereby forming a homopolymer duplex region,            and hybridizing the appended adaptor sequence of the            immobilized adaptor-target extension product to the adaptor            binding region of the immobilized circularization            oligonucleotide, thereby forming an immobilized looped            target extension product;        -   (g) cleaving the immobilized looped target extension product            (e.g., at the cleavable region) under a condition suitable            to release the homopolymer duplex region while retaining the            adaptor-hybridized region of the immobilized circularization            oligonucleotide;        -   (h) hybridizing the homopolymer region of the adaptor-target            extension product to the homopolymer region of the            immobilized circularization oligonucleotide thereby forming            an open circularized adaptor-target extension product with a            nick or gap;        -   (i) closing the gap and/or nick by conducting a gap-filling            primer extension reaction and/or a ligation reaction on the            open circularized adaptor-target extension product thereby            forming a closed circular target extension product which is            hybridized to the immobilized circularization            oligonucleotide having an adaptor binding region with a 3′            extendible end; and        -   (j) conducting a rolling circle amplification reaction using            the 3′ extendible end of the adaptor binding region under a            condition suitable to form an immobilized concatemer            molecule having tandem repeat regions comprising the            sequencing primer binding sequence, the target sequence, and            the spatial barcode sequence.    -   64. The method of embodiments 54-63, wherein a fluorescent image        of the support exhibits a contrast to noise ratio (CNR) of at        least 20.    -   65. The method of embodiments 54-64 wherein the at least one        hydrophilic polymer coating comprises a molecule selected from        the group consisting of polyethylene glycol (PEG), poly(vinyl        alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)        (PVP), poly(acrylic acid) (PAA), polyacrylamide,        poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)        (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),        poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),        polyglutamic acid (PGA), poly-lysine, poly-glucoside,        streptavidin, and dextran.    -   66. The method of embodiment 65, wherein the least one        hydrophilic polymer coating comprises polyethylene glycol (PEG).    -   67. The method of embodiments 54-66, wherein the substrate is        coated with a second hydrophilic polymer coating.    -   68. The method of embodiments 54-67, the at least one        hydrophilic polymer coating comprises a polymer having a        molecular weight of at least 1,000 Daltons.    -   69. The method of embodiments 54-68, wherein the at least one        hydrophilic polymer coating comprises a branched hydrophilic        polymer having at least 4 branches.    -   70. The method of embodiments 54-69, wherein the at least one        hydrophilic polymer coating comprises: (a) a first layer        comprising a first monolayer of polymer molecules tethered to        the surface; (b) a second layer comprising a second monolayer of        polymer molecules tethered to the first monolayer of polymer        molecules; and (c) a third layer comprising a third monolayer of        polymer molecules tethered to the second monolayer of polymer        molecules, wherein the polymer molecules of the first layer, the        second layer or the third layer comprises branched polymer        molecules.    -   71. The method of embodiments 54-70, wherein the support        comprises glass or plastic.    -   72. The method of embodiments 54-71, wherein the support        comprises a planar support or a bead.    -   73. The method of embodiments 54-72, wherein the target nucleic        acid molecule is derived from a biological sample that is placed        onto the plurality of capture oligonucleotides that are        immobilized on the low binding coating on the support.    -   74. The method of embodiments 54-73, wherein the target nucleic        acid molecule is hybridized (captured) on the support in a        manner that preserves spatial location information of the target        nucleic acid molecule in the biological sample.    -   75. The method of embodiments 54-74, wherein the condition which        is suitable for hybridizing at least a portion of the target        nucleic acid molecule to the target capture region of one of the        immobilized capture oligonucleotides comprises contacting the        low non-specific binding coating with a target nucleic acid        molecule and a hybridization buffer comprising:        -   (i) a first polar aprotic solvent having a dielectric            constant that is no greater than 40 and having a polarity            index of 4-9;        -   (ii) a second polar aprotic solvent having a dielectric            constant that is no greater than 115 and is present in the            hybridization buffer formulation in an amount effective to            denature double-stranded nucleic acids;        -   (iii) a pH buffer system that maintains the pH of the            hybridization buffer formulation in a range of about 4-8;            and        -   (iv) a crowding agent in an amount sufficient to enhance or            facilitate molecular crowding.    -   76. The method of embodiments 54-75, wherein the hybridization        buffer comprises the first polar aprotic solvent comprises        acetonitrile at 25-50% by volume of the hybridization buffer.    -   77. The method of embodiments 54-76, wherein the second polar        aprotic solvent comprises formamide at 5-10% by volume of the        hybridization buffer.    -   78. The method of embodiments 54-77, wherein the pH buffer        system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a        pH of 5-6.5    -   79. The method of embodiments 54-78, wherein the crowding agent        comprises polyethylene glycol (PEG) at 5-35% by volume of the        hybridization buffer.    -   80. The method of embodiments 54-79, wherein the hybridization        buffer further comprises betaine.    -   81. The method of embodiments 63-80, further comprising:        determining the sequence of the immobilized concatemer molecule        by:        -   (a) contacting the immobilized concatemer molecule with (i)            a plurality of polymerases, (ii) at least one            polymer-nucleotide conjugate comprising two or more            duplicates of a nucleotide moiety that are connected to a            core via a linker, and (iii) a plurality of sequencing            primers that hybridize with the sequencing primer binding            sequence, under a condition suitable for binding at least            one polymerase and at least one sequencing primer to a            portion of the immobilized concatemer molecule, and suitable            for binding at least one of the nucleotide moieties of the            polymer-nucleotide conjugate to the 3′ end of the sequencing            primer at a position that is opposite a complementary            nucleotide in the immobilized concatemer molecule wherein            the bound nucleotide moiety does not incorporate into the            sequencing primer;        -   (b) detecting and identifying the bound nucleotide moiety of            the polymer-nucleotide conjugate thereby determining the            sequence of the immobilized concatemer molecule;        -   (c) optionally repeating steps (a) and (b) at least once;        -   (d) contacting the immobilized concatemer molecule with (i)            a plurality of polymerases, and (ii) a plurality of            nucleotides, under a condition suitable binding at least one            polymerase to at least a portion of the immobilized            concatemer molecule and suitable for binding at least one of            the nucleotides from the plurality to the 3′ ends of the            hybridized sequencing primers at a position that is opposite            a complementary nucleotide in the immobilized concatemer            molecule wherein the bound nucleotides incorporate into the            hybridized sequencing primers; (e) optionally detecting the            incorporated nucleotides;        -   (f) optionally identifying the incorporation nucleotides            thereby determining or confirming the sequence of the            immobilized concatemer; and        -   (g) repeating steps (a)-(f) at least once. In some            embodiments, the determining the sequence of the immobilized            concatemer molecule comprises sequencing the target sequence            and the spatial barcode sequence.    -   82. The method of embodiments 63-80, further comprising:        determining the sequence of the immobilized concatemer molecule        by:        -   (a) contacting the immobilized concatemer molecule with (i)            a plurality of polymerases, (ii) a plurality of nucleotides,            and (iii) a plurality of sequencing primers that hybridize            with the sequencing primer binding sequence, under a            condition suitable for binding at least one polymerase and            at least one sequencing primer to a portion of the            immobilized concatemer molecule, and suitable for binding at            least one of the nucleotides to the 3′ end of the sequencing            primer at a position that is opposite a complementary            nucleotide in the immobilized concatemer molecule wherein            the bound nucleotide incorporates into the 3′ end of the            sequencing primer;        -   (b) detecting and identifying the incorporated nucleotide            thereby determining the sequence of the immobilized            concatemer molecule; and        -   (c) optionally repeating steps (a) and (b) at least once. In            some embodiments, the determining the sequence of the            immobilized concatemer molecule comprises sequencing the            target sequence and the spatial barcode sequence.    -   83. A method for nucleic acid sequence determination comprising:        -   (a) fixing a biological sample comprising a target nucleic            acid molecule to a surface of a substrate; and        -   (b) contacting said surface with a nucleotide moiety            comprising a detectable label under conditions sufficient to            allow a complex to be formed between said nucleotide moiety            and said target nucleic acid molecule, wherein an image of            said surface exhibits a contrast-to-noise ratio of greater            than or equal to about 5 when said image of said surface is            obtained using an inverted microscope and a camera under            non-signal saturating conditions while said surface is            immersed in a buffer and wherein said detectable label is a            fluorescent dye.    -   84. A method for nucleic acid sequence determination comprising:        -   (a) fixing a biological sample comprising a target nucleic            acid molecule to a surface of a substrate;        -   (b) contacting said surface with a polymer nucleotide            conjugate under conditions sufficient to allow a multivalent            binding complex to be formed between said polymer-nucleotide            conjugate and said target nucleic acid molecule, wherein            said polymer-nucleotide conjugate comprises a nucleotide and            a detectable label; and        -   (c) detecting said multivalent binding complex in the            presence of the biological sample fixed to said surface,            thereby determining an identity of said nucleotide in the            target nucleic acid molecule.

Further embodiments of the present disclosure are provided:

-   1. A method for analyzing biological molecules from a cellular    biological sample, wherein the cells of the cellular biological    sample comprise cellular nucleic acids and polypeptides, and wherein    at least one cell in the sample includes a target nucleic acid that    encodes a target polypeptide, the method comprising the general step    of:    -   a) providing a support comprising a low non-specific binding        coating to which a plurality of capture oligonucleotides and        optionally a plurality of circularization oligonucleotides are        immobilized, wherein the plurality of immobilized capture        oligonucleotides comprise (i) a target capture region that        hybridizes to at least a portion of a target nucleic acid        molecule, and (ii) a spatial barcode sequence, wherein the low        non-specific binding coating comprises at least one hydrophilic        polymer layer having a water contact angle of no more than 45        degrees;    -   b) contacting the low non-specific binding coating with the        cellular biological sample in the presence of a high efficiency        hybridization buffer under conditions suitable to promote        migration of the target nucleic acid molecule from the cellular        biological sample to one of the immobilized capture        oligonucleotides thereby forming an immobilized target nucleic        acid duplex, wherein the target nucleic acid molecule is        immobilized to the low non-specific binding coating in a manner        that preserves spatial location information of the target        nucleic acid molecule in the cellular biological sample;    -   c) conducting a primer extension reaction on the immobilized        target nucleic acid duplex thereby forming an immobilized target        extension product;    -   d) forming an open circular target molecule using the        immobilized circularization oligonucleotide, or if the low        non-specific binding coating does not already include an        immobilized circularization oligonucleotide then immobilizing a        soluble circularization oligonucleotide to the low non-specific        binding coating in proximity to the immobilized target extension        product and forming an open circular target molecule using the        now-immobilized circularization oligonucleotide;    -   e) forming a covalently closed circular target molecule which is        immobilized to the low non-specific binding coating;    -   f) conducting a rolling circle amplification reaction on the        immobilized covalently closed circular target molecule to form        an immobilized nucleic acid concatemer molecule having tandem        repeat regions comprising the target sequence and the spatial        barcode sequence; and    -   g) sequencing at least a portion of the nucleic acid concatemer,        including sequencing the target sequence and the spatial barcode        sequence, to determine the spatial location of the target        nucleic acid in the cellular biological sample.-   2. The method of embodiment 1, wherein step (g) comprises:    sequencing at least a portion of the nucleic acid concatemers using    an optical imaging system comprising a field-of-view (FOV) greater    than 1.0 mm².-   3. The method of embodiment 1, wherein the target nucleic acid    comprises RNA.-   4. The method of embodiment 3, wherein the spatial location of the    target RNA in the cellular biological sample corresponds to the    spatial location of at least one cell in the cellular biological    sample that expresses the target RNA which encodes the target    polypeptide.-   5. The method of embodiment 1, wherein the primer extension reaction    of step (c) comprises a reverse transcription reaction.-   6. The method of embodiment 1, wherein the high efficiency    hybridization buffer comprises:    -   (i) a first polar aprotic solvent having a dielectric constant        that is no greater than 40 and having a polarity index of 4-9;    -   (ii) a second polar aprotic solvent having a dielectric constant        that is no greater than 115 and is present in the high        efficiency hybridization buffer formulation in an amount        effective to denature double-stranded nucleic acids;    -   (iii) a pH buffer system that maintains the pH of the high        efficiency hybridization buffer formulation in a range of about        4-8; and    -   (iv) a crowding agent in an amount sufficient to enhance or        facilitate molecular crowding.-   7. The method of embodiment 6, wherein the high efficiency    hybridization buffer further comprises betaine.-   8. The method of embodiment 1, wherein the rolling circle    amplification reaction of step (g) comprises contacting the    covalently closed circularized target molecule (e.g., circularized    nucleic acid template molecule(s)) with a DNA polymerase, a    plurality of nucleotides, and at least one catalytic divalent    cation, under a condition suitable for generating at least one    nucleic acid concatemer, wherein the at least one catalytic divalent    cation comprises magnesium or manganese.-   9. The method of embodiment 1, wherein the rolling circle    amplification reaction of step (g) comprises:    -   a) contacting the covalently closed circularized target molecule        (e.g., circularized nucleic acid template molecule(s)) with a        DNA polymerase, a plurality of nucleotides, and at least one        non-catalytic divalent cation that does not promote        polymerase-catalyzed nucleotide incorporation into the 3′        extendible end, wherein the non-catalytic divalent cation        comprises strontium or barium; and    -   b) contacting the covalently closed circularized target molecule        with at least one catalytic divalent cation, under a condition        suitable for generating at least one nucleic acid concatemer,        wherein the at least one catalytic divalent cation comprises        magnesium or manganese.-   10. The method of embodiment 1, wherein the rolling circle    amplification reaction of step (f) can be conducted at a constant    temperature (e.g., isothermal) ranging from room temperature to    about 70° C.-   11. The method of embodiment 1, further comprising: conducting a    multiple displacement amplification (MDA) reaction prior to step    (g), wherein the MDA reaction comprises (1) contacting at least one    nucleic acid concatemer with at least one amplification primer    comprising a random sequence, a DNA polymerase having strand    displacement activity, a plurality of nucleotides, and a catalytic    divalent cation comprising magnesium or manganese, or wherein the    MDA reaction comprises (2) contacting at least one nucleic acid    concatemer with a DNA primase-polymerase enzyme, a DNA polymerase    having strand displacement activity, a plurality of nucleotides, and    a catalytic divalent cation comprising magnesium or manganese.-   12. The method of embodiment 9, further comprising, conducting the    following steps after the rolling circle amplification of step (f)    and prior to step (g):    -   a) forming a nucleic acid relaxant reaction mixture by        contacting the nucleic acid concatemer with one or a combination        of two or more compounds selected from a group consisting of        formamide, acetonitrile, ethanol, guanidine hydrochloride, urea,        potassium iodide and/or polyamines, to generate a relaxed        nucleic acid concatemer, wherein the forming a nucleic acid        relaxant reaction mixture is conducted with a temperature        ramp-up, a relaxant incubation temperature, and a temperature        ramp-down;    -   b) washing the relaxed concatemer;    -   c) forming a flexing amplification reaction mixture by        contacting the relaxed concatemer with a strand-displacing DNA        polymerase, a plurality of nucleotides, a catalytic divalent        cation, (in the absence of added amplification primers), to        generate double-stranded concatemers, wherein the forming a        flexing amplification reaction mixture is conducted with a        temperature ramp-up, a flexing incubation temperature, and a        temperature ramp-down;    -   d) washing the double-stranded concatemer; and    -   e) repeating steps (a)-(d) at least once.-   13. The method of embodiment 1, wherein the sequencing of step (g)    comprises monitoring the sequential binding of labeled nucleotides    in a template strand of the concatemer (e.g., sequencing by    binding).-   14. The method of embodiment 13, wherein the sequencing of step (g)    further comprises monitoring the incorporation of the labeled    nucleotides in a template strand of the concatemer (e.g., sequencing    by synthesis).-   15. The method of embodiment 1, wherein the sequencing of step (g)    comprises detecting a complex formed between a polymerase and a    primed template strand of the concatemer, wherein the polymerase is    optionally labeled.-   16. The method of embodiment 1, wherein the sequencing of step (g)    comprises: contacting the plurality of nucleic acid concatemers with    a plurality of sequencing primers, a plurality of polymerases, and a    plurality of multivalent molecules, wherein each of the multivalent    molecules comprise two or more duplicates of a nucleotide moiety    that are connected to a core via a linker.-   17. The method of embodiment 16, wherein the multivalent molecule    comprises:    -   a) a core, and    -   b) a plurality of nucleotide arms which comprise (i) a core        attachment moiety, (ii) a spacer comprising a PEG moiety, (iii)        a linker, and (iv) a nucleotide unit, wherein the core is        attached to the plurality of nucleotide arms, wherein the spacer        is attached to the linker, wherein the linker is attached to the        nucleotide unit, wherein the nucleotide unit comprises a base,        sugar and at least one phosphate group, and wherein the linker        is attached to the nucleotide unit through the base, wherein the        linker comprises an aliphatic chain or an oligo ethylene glycol        chain where both linker chains having 2-6 subunits and        optionally the linker includes an aromatic moiety.-   18. The method of embodiment 16, wherein the multivalent molecule    comprises a core attached to multiple nucleotide arms, and wherein    the multiple nucleotide arms have the same type of nucleotide unit    which is selected from a group consisting of dATP, dGTP, dCTP, dTTP    and dUTP.-   19. The method of embodiment 16, wherein the multivalent molecule    further comprises a plurality of multivalent molecules which    includes a mixture of multivalent molecules having two or more    different types of nucleotides selected from a group consisting of    dATP, dGTP, dCTP, dTTP and dUTP.-   20. The method of embodiment 16, wherein the multivalent molecule    comprises a core attached to multiple nucleotide arms, wherein the    core is labeled with detectable reporter moiety.-   21. The method of embodiment 16, wherein the detectable reporter    moiety comprises a fluorophore.-   22. The method of embodiment 16, wherein the core comprises an    avidin-like moiety and the core attachment moiety comprises biotin.-   23. The method of embodiment 1, wherein the sequencing of step (h)    comprises:    -   a) contacting the plurality of nucleic acid concatemers with (i)        a plurality of polymerases, (ii) at least one multivalent        molecule comprising two or more duplicates of a nucleotide        moiety that are connected to a core via a linker, and (iii) a        plurality of sequencing primers that hybridize with a portion of        the concatemers, under a condition suitable for binding at least        one polymerase and at least one sequencing primer to a portion        of one of the nucleic acid concatemer molecules, and suitable        for binding at least one of the nucleotide moieties of the        multivalent molecule to the 3′ end of the sequencing primer at a        position that is opposite a complementary nucleotide in the        concatemer molecule wherein the bound nucleotide moiety does not        incorporate into the sequencing primer;    -   b) detecting and identifying the bound nucleotide moiety of the        multivalent molecule thereby determining the sequence of the        concatemer molecule;    -   c) optionally repeating steps (a) and (b) at least once;    -   d) contacting the concatemer molecule with (i) a plurality of        polymerases, and (ii) a plurality of nucleotides, under a        condition suitable binding at least one polymerase to at least a        portion of the concatemer molecule and suitable for binding at        least one of the nucleotides from the plurality to the 3′ ends        of the hybridized sequencing primers at a position that is        opposite a complementary nucleotide in the concatemer molecule        wherein the bound nucleotides incorporate into the hybridized        sequencing primers;    -   e) optionally detecting the incorporated nucleotides;    -   f) optionally identifying the incorporation nucleotides thereby        determining or confirming the sequence of the concatemer; and    -   g) repeating steps (a)-(f) at least once.

III. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: In Situ Sequencing

A. Preparing Tissue Samples

Fresh frozen tissue samples from an animal or human subject is embeddedin paraffin or OCT (Optimal Cutting Temperature) and cryo-sectioned atapproximately 10 micron thickness. The embedded tissue slices arepositioned on a support that lacks capture oligonucleotides. Forexample, the tissue slices are positioned on a slide (e.g., SuperFrostPlus microscope slide, e.g., from Fisher Scientific catalog No.12-550-15) and stored at −80° C. until ready for use.

The slides are removed from −80° C. and thawed to room temperature. Thetissue sample is fixed by applying to the tissue slices 3% (w/v)paraformaldehyde in DEPC-PBS and incubated for approximately 5 minutesat room temperature. The tissue sections are rinsed at least twice withDEPC-PBS. The tissue is permeabilized with acidic condition by dippingthe slide into a solution of 0.1 M HCl at room temperature forapproximately 5 minutes. The slides are washed with DEPC-PBS at roomtemperature for at least 1 minute. The slides are dehydrated in anethanol series: (1) 70% ethanol at room temperature for approximately 1minute; (2) 100% ethanol at room temperature for approximately 1 minute.The slides are air-dried. Hybridization chambers are mounted over thetissue slices on the slide with SECURESEAL hybridization chambers (e.g.,from Grace Bio-Labs). The tissue sections are rehydrated with DEPC-PBS-Tand then with DEPC-PBS.

B. In Situ Reverse Transcriptase Reaction

A reverse transcriptase reaction is prepared by adding to the chamber: areverse transcriptase enzyme (e.g., TranscriptME Reverse Transcriptasewhich is an M-MuLV reverse transcriptase from CytoGen), reversetranscriptase buffer, dNTPs (500 uM), random primers (e.g., decamers, 5uM), RNase inhibitor (1 U/uL), BSA (0.2 ug/uL), and DEPC-water. Anexemplary reverse transcriptase buffer can include: 50 mM Tris-HCl (pH8.3); 75 mM KCl; 3 mM MgCl₂; and 10 mM DTT. The chamber is sealed, andthe slide is placed in a humidity chamber, and incubated at 37° C. forat least 6 hours. The reverse transcriptase reagents are removed. Thetissue slices are fixed with 3% (w/v) paraformaldehyde at roomtemperature for approximately 30 minutes. The tissue slices are washedseveral times with DPEC-PBS-T.

C. Rolling Circle Amplification

The padlock probes are 70-100 nucleotides in length and arephosphorylated at their 5′ ends, and include: terminal target regions(5′ arm and 3′ arm) that hybridize to the target sequence each 15nucleotides in length, backbone region that includes an ID sequence(about 6-20 nucleotides in length) and optionally an anchor bindingsequence (about 6-20 nucleotides in length). In some embodiments, theterminal target regions of the padlock probes comprise random sequences.Padlock probe hybridization and ligation is conducted in situ by addingto the tissue slices: the padlock probe (e.g., approximately 10 nM ofeach type of padlock probe), 1×Tth ligase buffer, KCl (0.05 M),formamide (20%), BSA (0.2 ug/uL), Tth ligase enzyme (0.5 U/uL) andRNaseH (0.4 U/uL). An exemplary ligase buffer includes 20 mM Tris-HCl(pH 8.3), 25 mM KCl, 10 mM MgCl₂, 0.5 mM NAD, and 0.01% Triton X-100.The padlock probe hybridization reaction was conducted at about 37° C.for approximately 30 minutes, then at about 45° C. for about 1½ to 2hours. The slide is washed several times with DEPC-PBS-T.

A two-step rolling circle amplification is conducted to generateconcatemers in the cells of the tissue slices.

Step One: add to the tissue slices a non-catalytic solution: 10 mM ACESpH 7.4, dNTPs (10 uM), 1 mM strontium acetate, 0.01% Tween-20, 50 mMammonium sulfate, and 10 mM DTT. The chamber is sealed and incubated ina humidity chamber at room temperature or 35° C. for 15 minutes. Thenon-catalytic solution is removed from the chamber.

Step Two: add to the tissue slices a catalytic solution: 50 mM ACES pH7.4, 100 mM potassium acetate, 10 mM MgSO₄, dNTPs (2 mM), 10 mM DTT,0.01% Tween-20, 50 mM ammonium sulfate, and 10 mM DTT. Optionally,compaction oligonucleotides are included at 10-200 nM. The chamber issealed and placed in a humidity chamber. Rolling circle amplificationreaction is conducted at room temperature or 35° C. for different timeranges from 5 minutes up to 2 hours. The tissue slices are washed with abuffer containing 50 mM Tris-HCl pH 8, 750 mM NaCl, 0.1 mM EDTA and0.02% Tween-20.

D1. Multiple Displacement Amplification with Soluble Random Primers

Multiple displacement amplification (MDA) is conducted following therolling circle amplification reaction to generate branched concatemers.The MDA reaction is conducted by adding to the tissue slices: 50 mM TrispH7.5, 75 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 2.5% glycerol, 0.1 mg/mL BSA,1.5-2 mM dNTPs, 1-10 uM random-sequence hexamers (exonucleaseresistant), and a strand displacing DNA polymerase. The stranddisplacing DNA polymerase that are tested include phi29 (wild type),EquiPhi29 (e.g., Thermo Fisher Scientific, catalog No. A39390), QualiPhi(e.g., from 4basebio, catalog No. 510025), large fragment of Bst DNApolymerase exonuclease minus (e.g., Lucigen, catalog NO. 30027-1), andlarge fragment of Bsu DNA polymerase exonuclease minus (e.g., NewEngland Biolabs, catalog No. MS330S). The DNA polymerases are typicallyadded at 150 nM. Alternative MDA formulas can include:commercially-available buffers including: phi29 10× reaction buffer(Thermo Fisher, catalog No. B62) supplemented with 1-20 mM DTT and 0.5-4mM dNTPs; EquiPhi29 10× reaction buffer (Thermo Fisher Scientific,catalog No. B39) supplemented with 1-20 mM DTT and 0.5-4 mM dNTPs; andTruePrime Kit buffer (e.g., Lucigen, catalog No. SYG370025) supplementedwith 0.5-4 mM dNTPs. The chamber is place in a humidity chamber toconduct the multiple displacement amplification reaction. Differentincubation conditions are tested including: temperatures ranging from30-45° C., for 30-90 minutes. The chamber is washed with a buffercontaining either (1) 50 mM Tris-HCl pH 8, 750 mM NaCl, 0.1 mM EDTA,0.02% Tween-20; or (2) 3×SSC buffer, followed by a buffer containing 50mM Tris pH 8, 100 mM NaCl, 0.1 mM EDTA, and 0.01% Tween-20.

D2. Multiple Displacement Amplification with DNA Primase-Polymerase

Following the rolling circle amplification reaction, an alternative MDAreaction is conducted using DNA primase-polymerase and lacking anyprimers to generate branched concatemers. Different MDA formulas aretested. One of the MDA formulas contains 50 mM Tris pH7.5, 75 mM NaCl,10 mM MgCl₂, 1 mM DTT, 2.5% glycerol, 0.1 mg/mL BSA, 1.5-2 mM dNTPs, astrand displacing DNA polymerase, and DNA primase-polymerase. Other MDAformulas can include commercially-available buffers including: phi29 10×reaction buffer (Thermo Fisher, catalog No. B62) supplemented with 1-20mM DTT and 0.5-4 mM dNTPs; EquiPhi29 10× reaction buffer (Thermo FisherScientific, catalog No. B39) supplemented with 1-20 mM DTT and 0.5-4 mMdNTPs; and TruePrime Kit buffer (e.g., Lucigen, catalog No. SYG370025)supplemented with 0.5-4 mM dNTPs. The strand displacing DNA polymerasethat are tested include phi29 (wild type), EquiPhi29 (e.g., ThermoFisher Scientific, catalog No. A39390), QualiPhi (e.g., from 4basebio,catalog No. 510025), large fragment of Bst DNA polymerase exonucleaseminus (e.g., Lucigen, catalog NO. 30027-1), and large fragment of BsuDNA polymerase exonuclease minus (e.g., New England Biolabs, catalog No.MS330S). The DNA polymerases were typically added at 150 nM. The DNAprimase-polymerase enzyme was Tth PrimPol (4basebio, catalog No.390100). The chamber is place in a humidity chamber to conduct themultiple displacement amplification reaction. Different incubationconditions are tested including: temperatures ranging from 30−45° C.,for 30-90 minutes. The chamber is washed with a buffer containing either(1) 50 mM Tris-HCl pH 8, 750 mM NaCl, 0.1 mM EDTA, 0.02% Tween-20; or(2) 3×SSC buffer, followed by a buffer containing 50 mM Tris pH 8, 100mM NaCl, 0.1 mM EDTA, and 0.01% Tween-20.

D3. Relaxant Conditions and Flexing Amplification

Instead of conducting a multiple displacement amplification reactionfollowing rolling circle amplification, the tissue slices are subject toa relaxant condition followed by a flexing amplification reaction togenerate highly compact concatemers.

A buffer containing nucleic acid relaxing agents is deposited onto thetissue slices with (1) a temperature ramp-up, incubation, andtemperature ramp-down profile, followed by (2) a flexing amplificationreaction using a strand-displacing DNA polymerase. Multiple cycles ofstages (1) and (2) were tested.

Relaxant Conditions:

Different relaxing buffer formulas are tested. Exemplary relaxing agentscan include nucleic acid denaturants, chaotropic compounds, amidecompounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents caninclude sodium iodide, potassium iodide and polyamines

The relaxant reaction mixture can contain any one or a combination oftwo or more of a group selected from urea, guanidine hydrochloride,guanidine thiocyanate, formamide, acetamide, NN-dimethylformamide (DMF),acetonitrile, DMSO (dimethyl sulfoxide), 1,4-dioxane, tetrahydrofuran,1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol,glycerol, 1,2-dimethyoxyethane, 2-methoxyethanol, sodium iodide,potassium iodide and/or polyamines.

Different nucleic acid relaxing temperature profiles are tested in ahumidity chamber. Typically, a nucleic acid relaxing temperature profilecan include: T1 initial temperature; T2 temperature ramp-up; incubatefor the nucleic acid relaxing reaction; and T3 temperature ramp-down.

An exemplary nucleic acid relaxing temperature cycle profile caninclude: T1 initial temperature is 25° C.; T2 ramp-up to 55° C. with atemperature gradient of +1° C./second; incubate at 55° C. for 30seconds; T3 ramp-down to 25° C. with a temperature gradient of −1°C./second.

After the T3 ramp-down, the chamber is washed to remove the relaxingbuffer. The Wash buffer contained 1×SSC and 0.1 mM cobalt hexamine.

Flexing Amplification:

A buffer containing a strand-displacing DNA polymerase is deposited ontothe tissue slices to conduct flexing amplification with amplificationtemperature cycling.

Any strand-displacing DNA polymerase can be used, including largefragment of Bst DNA polymerase (e.g., exonuclease minus), phi29 DNApolymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNApolymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase,M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or DeepVent DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNApolymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

For example, large fragment of Bst DNA polymerase is tested. A flexingamplification reaction buffer is deposited onto the tissue slices: BstDNA polymerase (400 nM), 20 mM Tris pH 8.5, 50 mM KCl, 5 mM MgSO₄, 0.1%Tween-20, 1.5 M Betaine, and 0.25 mM dNTP (total).

Different flexing amplification temperature cycle profiles can be testedin a humidity chamber. For example, a single flexing amplificationtemperature cycle profile can include: T1 initial temperature, T2temperature ramp-up, incubate for the flexing amplification reaction, T3temperature ramp-down. The temperature cycles can be repeated 2-50 timesor more.

An exemplary flexing amplification temperature cycle profile caninclude: T1 initial temperature is 25° C.; T2 ramp-up to 63° C. with atemperature gradient of +1° C./second; incubate at 63° C. for 55seconds; T3 ramp-down to 25° C. with a temperature gradient of −1°C./second. The relaxing stage and flexing amplification stage representa cycle. The cycles can be repeated 5-15 times.

After the last flexing T3 ramp-down, the chamber is washed to remove therelaxing buffer. The Wash buffer can contain 1×SSC and 0.1 mM cobalthexamine.

E. Sequencing with Multivalent Molecules

Multivalent molecules comprising a fluorescently-labeled streptavidincore attached to multiple nucleotide arms (see FIGS. 5A, 5B and 5D) areused to sequence the concatemers in the cells of the tissue slices. Anon-catalytic buffer can be flowed onto the tissue slices, where thenon-catalytic buffer includes 20 nM Klenow polymerase (or other suitablepolymerase), sequencing primers, 2.5 mM strontium and labeledmultivalent molecules (e.g., at 2.5 uM). A fluorescent image of thepolymerase bound to the labeled multivalent molecule (ternary complexwhere the multivalent molecule is not incorporated) can be obtained. Themultivalent molecule can be dissociated by adding a wash buffer having10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% Triton X100 (butlacking strontium). A catalytic buffer can be flowed onto the tissueslices, where the catalytic buffer can include 20 nM Klenow polymerase(or other suitable polymerase), magnesium, optionally sequencingprimers, and labeled or non-labeled nucleotides. The nucleotides canhave 2′ or 3′ chain terminating moiety, such as for example an azide,azido or azido-methyl group. The nucleotides can incorporate into thesequencing primers to extend the primers. If the incorporated nucleotideis labeled, an image can be obtained. The chain-terminating moiety inthe incorporated nucleotides can be removed using an appropriate reagent(e.g., phosphine compound). Repeat cycles can be conducted, whichinclude non-catalytic binding with the multivalent molecules, imagingthe bound multivalent molecules, catalytic incorporation of nucleotides,and optional imaging the incorporated nucleotides.

Example 2: In Situ Single Cell Sequencing

A single cell can be obtained from an animal or human and can be placedin simple or complex cell media for at least 15 minutes. The simple cellmedia can be PBS (phosphate buffered saline), DPBS (Dulbecco'sphosphate-buffered saline), HBSS (Hank's balanced salt solution), DMEM(Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum EssentialMedium), and/or EBSS. The complex cell media can be fetal bovine serum,blood plasma or blood serum.

The single cell can be embedded in paraffin or OCT (Optimal CuttingTemperature) and cryo-sectioned as described in Example 1-A above. Thesections can be fixed as described in Example 1-A above. The sections ofthe single cell can be positioned on a glass support that is passivatedwith a low non-specific binding coating and lacks immobilized captureoligonucleotides. The sectioned single cell, while being positioned onthe passivated support, can be permeabilized, dehydrated and rehydratedas described in Example 1-A above.

The sections of the single cell can be subjected to reversetranscriptase as described in Example 1-B above.

The sections of the single cell can be subject to rolling circleamplification, to generate concatemers, as described in Example 1-Cabove.

Following rolling circle amplification, the sections of the single cellcan be subjected to multiple displacement amplification using randomprimers, to generate branched concatemers, as described in Example 1-D1above, or can be subjected to multiple displacement amplification usingDNA primase-polymerase to generate branched concatemers, as described inExample 1-D2 above. Alternatively, following rolling circleamplification, the sections of the single cell can be subjected to arelaxant condition and flexing amplification, to generate highly compactconcatemers, as described in Example 1-D3 above.

The concatemers can be sequenced using multivalent molecules asdescribed in Example 1-E above.

Example 3: Biological Molecule Capture on a Low Non-Specific BindingCoating

A. Preparing Tissue Samples

Fresh frozen tissue samples from an animal or human subject is embeddedin paraffin or OCT (Optimal Cutting Temperature) and cryo-sectioned atapproximately 10 micron thickness. The tissue slices are positioned on asupport which is passivated with a low non-specific binding coatingwhich includes capture oligonucleotides immobilized to the coating. Thecoating optionally also includes immobilized circularizationoligonucleotides (FIG. 2). The tissue slices on the support can bestored at −80° C. until ready for use.

The low non-specific binding coating can have an array of surfacefeatures (e.g., shaped as spots, FIG. 3), where the features each haveimmobilized thereon approximately 100,000 or more captureoligonucleotides. The capture oligonucleotides include a target captureregion, a spatial barcode sequence (e.g., FIG. 2) and optionally asequencing primer binding sequence. Different features contain captureoligonucleotides with different spatial barcode sequences. Differentfeatures contain capture oligonucleotides with the same or differenttarget capture region sequences.

The slides are removed from −80° C. and thawed to room temperature. Thetissue sample is fixed by applying to the tissue slices 3% (w/v)paraformaldehyde in DEPC-PBS and incubated for approximately 5 minutesat room temperature.

B. Surface Capture of Target Nucleic Acids

The tissue sections are rinsed at least twice with DEPC-PBS. The cellsin the tissue slices are permeabilized by flowing an acidic solution of0.1 M HCl on the tissue slices at room temperature. A high efficiencyhybridization solution is flowed onto the tissue slices to allow thenucleic acids from the tissue to migrate from the tissue to the captureoligonucleotides on the passivated support. The high efficiencyhybridization solution includes: (i) acetonitrile at 25-50% by volume ofthe high efficiency high efficiency hybridization buffer; (ii) formamideat 5-10% by volume of the high efficiency high efficiency hybridizationbuffer; (iii) 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) polyethylene glycol (PEG) (e.g., PEG 4000) at 5-35% byvolume of the high efficiency high efficiency hybridization buffer. Thetissue slices are incubated at approximately 55° C. for about 3 minutes,and then at 37° C. for about 3 minutes, then at room temperature forabout 3 minutes. The tissue slices are washed with DEPC-PBS at roomtemperature.

C. Reverse Transcriptase Reaction

A reverse transcriptase reaction was prepared by adding to the tissueslices: a reverse transcriptase enzyme (e.g., TranscriptME ReverseTranscriptase which is an M-MuLV reverse transcriptase from CytoGen),reverse transcriptase buffer, dNTPs (500 uM), 5 uM reverse transcriptionprimers (e.g., target-specific primers or random-sequence decamers),RNase inhibitor (1 U/uL), BSA (0.2 ug/uL), and DEPC-water. An exemplaryreverse transcriptase buffer can include: 50 mM Tris-HCl (pH 8.3); 75 mMKCl; 3 mM MgCl₂; and 10 mM DTT. The tissue slices are placed in ahumidity chamber, and incubated at 37° C. for at least 6 hours orovernight. The reverse transcriptase reagents are removed by washingwith DEPC-PBS at room temperature. The tissue slices are fixed with 3%(w/v) paraformaldehyde at room temperature for approximately 30 minutes.The tissue slices are washed several times with DPEC-PBS-T.

The cells of the tissue slices are enzymatically removed withcollagenase, neutral dispase protease and/or thermolysin (e.g.,LIBERASE) enzymes.

D. Rolling Circle Amplification

A two-step rolling circle amplification is conducted to generateconcatemers in the cells of the tissue slices.

Step One: add to the tissue slices a non-catalytic solution: 10 mM ACESpH 7.4, dNTPs (10 uM), 1 mM strontium acetate, 0.01% Tween-20, 50 mMammonium sulfate, and 10 mM DTT. The chamber is sealed and incubated ina humidity chamber at room temperature or 35° C. for 15 minutes. Thenon-catalytic solution is removed from the chamber.

Step Two: add to the tissue slices a catalytic solution: 50 mM ACES pH7.4, 100 mM potassium acetate, 10 mM MgSO₄, dNTPs (2 mM), 10 mM DTT,0.01% Tween-20, 50 mM ammonium sulfate, and 10 mM DTT. Optionally,compaction oligonucleotides are included at 10-200 nM. The chamber issealed and placed in a humidity chamber. Rolling circle amplificationreaction is conducted at room temperature or 35° C. for different timeranges from 5 minutes up to 2 hours. The tissue slices are washed with abuffer containing 50 mM Tris-HCl pH 8, 750 mM NaCl, 0.1 mM EDTA and0.02% Tween-20.

E1. Multiple Displacement Amplification with Soluble Random Primers

Multiple displacement amplification (MDA) is conducted following therolling circle amplification reaction to generate branched concatemersusing the protocol described in Example 1-D1 above.

E2. Multiple Displacement Amplification with DNA Primase-Polymerase

Following the rolling circle amplification reaction, an alternative MDAreaction is conducted using DNA primase-polymerase and lacking anyprimers to generate branched concatemers, using the protocol describedin Example 1-D2 above.

E3. Relaxant Conditions and Flexing Amplification

Instead of conducting a multiple displacement amplification reactionfollowing rolling circle amplification, the tissue slices are subject toa relaxant condition followed by a flexing amplification reaction togenerate highly compact concatemers, using the protocol described inExample 1-D3 above.

F. Sequencing with Multivalent Molecules

The concatemers were sequenced using multivalent molecules andnucleotides as described in Example 1-E above.

Example 4: Capturing Nucleic Acids from a Single Cell onto a on a LowNon-Specific Binding Coating

A single cell can be obtained from an animal or human and can be placedin simple or complex cell media for at least 15 minutes. The simple cellmedia can be PBS (phosphate buffered saline), DPBS (Dulbecco'sphosphate-buffered saline), HBSS (Hank's balanced salt solution), DMEM(Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum EssentialMedium), and/or EBSS. The complex cell media can be fetal bovine serum,blood plasma or blood serum.

The single cell can be embedded in paraffin or OCT (Optimal CuttingTemperature) and cryo-sectioned as described in Example 1-A above. Thesections can be fixed as described in Example 1-A above.

The tissue slices are positioned on a support which is passivated with alow non-specific binding coating which includes capture oligonucleotidesimmobilized to the coating. The coating optionally also includesimmobilized circularization oligonucleotides (FIG. 2). The tissue sliceson the support can be stored at −80° C. until ready for use.

The low non-specific binding coating can have an array of surfacefeatures (e.g., shaped as spots, FIG. 3), where the features each haveimmobilized thereon approximately 100,000 or more captureoligonucleotides. The capture oligonucleotides include a target captureregion, a spatial barcode sequence (e.g., FIG. 2) and optionally asequencing primer binding sequence. Different features contain captureoligonucleotides with different spatial barcode sequences. Differentfeatures contain capture oligonucleotides with the same or differenttarget capture region sequences.

The slides are removed from −80° C. and thawed to room temperature. Thetissue sample is fixed by applying to the tissue slices 3% (w/v)paraformaldehyde in DEPC-PBS and incubated for approximately 5 minutesat room temperature.

The nucleic acids (e.g., RNA) from the embedded single cell can becaptured by the immobilized capture oligonucleotides on the coating,using the high efficiency hybridization solution as described in Example3-B above.

The captured RNA can be subjected to a reverse transcription reaction togenerate cDNA, followed by fixation and enzymatic cell removal, asdescribed in Example 3-C above.

The cDNA can be subjected to the two-step rolling circle amplificationreaction as described in Example 3-D above.

Following rolling circle amplification, the sections of the single cellcan be subjected to multiple displacement amplification using randomprimers, to generate branched concatemers, as described in Example 3-E1above, or can be subjected to multiple displacement amplification usingDNA primase-polymerase to generate branched concatemers, as described inExample 3-E2 above. Alternatively, following rolling circleamplification, the sections of the single cell can be subjected to arelaxant condition and flexing amplification, to generate highly compactconcatemers, as described in Example 3-E3 above.

The concatemers can be sequenced using multivalent molecules asdescribed in Example 3-F above.

Example 5: Design Specifications for a Fluorescence Imaging Module forGenomics Applications

A non-limiting example of design specifications for a fluorescenceimaging module of the present disclosure is provided in Table 1.

Design Parameter Specification Numerical aperture ≥0.3 Image qualityDiffraction limited Field-of-view (FOV) >2.0 mm² Image plane curvatureBest focal plane within 100 nm for >90% of the FOV, within 150 nm for99% of the FOV, and within 200 nm for the entire FOV Image distortion<0.5% across the FOV Magnification 2× to 20× Camera pixel size at sampleplane ≥2 × optical system modulation transfer function (MTF) limitCoverslip thickness >700 μm Number of fluorescence imaging ≥3 channelsChromatic focal plane difference at ≤100 nm equivalent at sample camerabetween all imaging channels plane Number of AF channels 1 Imaging time≤2 seconds per FOV Autofocus Single step autofocus with error correctionAutofocus accuracy <100 nm Scanning stage step and settle time <0.4seconds Channel-specific optimized tube lens 1 per imaging channelIllumination optical path Liquid light guide with underfilled entranceaperture

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for analyzing a target nucleic acidsequence in situ, the method comprising: (a) providing a cell or atissue comprising a plurality of nucleic acid molecules, wherein saidplurality of nucleic acid molecules comprises said target nucleic acidsequence; (b) contacting a subset of said plurality of nucleic acidmolecules with a detectable nucleotide conjugate under conditionssuitable to form a binding complex between (i) at least two nucleotidemoieties of said detectable nucleotide conjugate and (ii) at least onenucleotide of each of at least two of said target nucleic acid sequenceof said plurality of nucleic acid molecules within said cell or saidtissue, wherein said subset of said plurality of nucleic acid moleculesis primed; (c) detecting said binding complex; and (d) performing (b) to(c) for at least two other nucleotides of said at least two of saidtarget nucleic acid sequence of said plurality of nucleic acidmolecules, thereby identifying said target nucleic acid sequence.
 2. Themethod of claim 1, wherein said cell or said tissue is immobilized to aninterior surface of a flow cell.
 3. The method of claim 2, wherein saidinterior surface of said flow cell comprises one or more hydrophilicpolymer layers.
 4. The method of claim 3, wherein said one or morehydrophilic polymer layers comprises a polymer comprising polyethyleneglycol (PEG).
 5. The method of claim 4, wherein said one or morehydrophilic polymer layers comprises a branched polymer.
 6. The methodof claim 3, wherein said one or more hydrophilic polymer layerscomprises a branched polymer.
 7. The method of claim 2, wherein saidinterior surface has a water contact angle comprising less than or equalto 45 degrees.
 8. The method of claim 1, further comprisingpermeabilizing said tissue or lysing said cell prior to said contactingin (b).
 9. The method of claim 2, wherein an image of said interiorsurface exhibits a contrast-to-noise ratio (CNR) of greater than orequal to about 10 when said CNR is measured by: (a) contacting saidinterior surface with a fluorescently labeled nucleotide moleculecomprising a nucleic acid sequence that is complementary to at least aportion of a capture oligonucleotide immobilized to said interiorsurface; and (b) following (a), imaging said interior surface using aninverted microscope and a camera under non-signal saturating conditionswhile said interior surface is immersed in a buffer.
 10. The method ofclaim 1, wherein said identifying said target nucleic acid sequence in(d) is performed with an accuracy of base-calling that is characterizedby a Q-score of greater than 25 for at least 80% of nucleotidesidentified.
 11. The method of claim 1, further comprising determining aspatial location of said target nucleic acid sequence within said cellor said tissue.
 12. The method of claim 1, further comprisingdetermining a cell type of said cell based, at least in part, on saididentifying said target nucleic acid sequence in (d).
 13. The method ofclaim 1, further comprising determining a tissue type of said tissuebased, at least in part, on said identifying said target nucleic acidsequence in (d).
 14. The method of claim 1, wherein said detectablenucleotide conjugate comprises: (a) a common core; and (b) said at leasttwo nucleotide moieties coupled to said common core, wherein said commoncore comprises a polymer, a micelle, a liposome, a microparticle, ananoparticle, or a quantum dot.
 15. The method of claim 14, wherein saidcommon core is spheroidal.
 16. The method of claim 14, wherein saiddetectable nucleotide conjugate further comprises a detectable moiety.17. The method of claim 16, wherein said detectable moiety is coupled tosaid common core.
 18. The method of claim 1, wherein in (b) saiddetectable nucleotide conjugate is included in a mixture of a pluralityof detectable nucleotide conjugates, wherein each of said plurality ofdetectable nucleotide conjugates comprises different types of nucleotidemoieties from each other.
 19. The method of claim 18, wherein saiddifferent types of said nucleotide moieties comprise at least threedifferent types of said nucleotide moieties.
 20. The method of claim 1,wherein said at least two nucleotide moieties do not comprise a blockinggroup coupled thereto.
 21. The method of claim 1, wherein said pluralityof nucleic acid molecules comprises a blocking group sufficient toprevent incorporation of said at least two nucleotide moieties into saidat least two of said target nucleic acid sequence.
 22. The method ofclaim 1, wherein said tissue is a cancerous tissue.
 23. The method ofclaim 1, wherein said cell is a cancer cell.
 24. The method of claim 1,wherein said detecting said binding complex comprises imaging said cellor said tissue us in one or more image sensors.
 25. The method of claim24, wherein said one or more image sensors comprises a photodetectorarray.
 26. The method of claim 1, further comprising washing said cellor said tissue following (c) to remove said detectable nucleotideconjugate from said cell or said tissue.